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Despite early optimism, therapeutics targeting oxidative phosphorylation (OxPhos) have faced clinical setbacks, stemming from their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to cancerous mitochondria inside acute myeloid leukemia (AML) cells. Unlike healthy cells which couple respiration to the synthesis of ATP, AML mitochondria were discovered to support inner membrane polarization by consuming ATP. Because matrix ATP consumption allows cells to survive bioenergetic stress, we hypothesized that AML cells may resist cell death induced by OxPhos damaging chemotherapy by reversing the ATP synthase reaction. In support of this, targeted inhibition of BCL-2 with venetoclax abolished OxPhos flux without impacting mitochondrial membrane potential. In surviving AML cells, sustained polarization of the mitochondrial inner membrane was dependent on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to downregulations in both the proton-pumping respiratory complexes, as well as the endogenous F 1 -ATPase inhibitor ATP5IF1 . In treatment-naive AML, ATP5IF1 knockdown was sufficient to drive venetoclax resistance, while ATP5IF1 overexpression impaired F 1 -ATPase activity and heightened sensitivity to venetoclax. Collectively, our data identify matrix ATP consumption as a cancer-cell intrinsic bioenergetic vulnerability actionable in the context of mitochondrial damaging chemotherapy., Competing Interests: ADS has received research funding from Takeda Pharmaceuticals and BMS, and consulting fees/honorarium from Takeda, Astra Zeneca, BMS and Novartis. ADS is named on a patent application for the use of DNT cells to treat AML. ADS is a member of the Medical and Scientific Advisory Board of the Leukemia and Lymphoma Society of Canada and the Therapy Acceleration Program for the Leukemia and Lymphoma Society. TPL has received Scientific Advisory Board membership, consultancy fees, honoraria, and/or stock options from Keystone Nano, Flagship Labs 86, Dren Bio, Recludix Pharma, Kymera Therapeutics, and Prime Genomics. DJF has received research funding, honoraria, and/or stock options from AstraZeneca, Dren Bio, Recludix Pharma, and Kymera Therapeutics.

Oxidation and assimilation of persulfides in bacteria is often catalyzed by a persulfide dioxygenase and sulfurtransferase in consecutive reactions. Enzymes responsible for the oxidation of persulfides have not been clearly defined in Pseudomonas aeruginosa PAO1. The characterized mercaptopropionate dioxygenase (MDO) in P. aeruginosa PAO1 has been proposed to catalyze the oxidation of 3-mercaptopropionate. However, the physiological role of MDO is uncertain given the expression of a sulfurtransferase (ST) enzyme on the same operon as the thiol dioxygenase. The st gene had a co-occurrence frequency with mdo of 0.94 demonstrating the co-expression and physiological link of the two genes. There are four tandem rhodanese domains in the ST enzyme with two of the domains containing potential catalytic Cys residues (Cys191 and Cys435) capable of forming a persulfide. Only Cys435 was accessible in thiol quantification assays, and results from H/D-X MS analyses further established the accessibility of the domain containing Cys435. Both thiosulfate and mercaptopyruvate served as sulfur donors to the ST enzyme, with Cys435 forming the persulfide intermediate. Kinetic investigations of MDO suggested the enzyme had a broader substrate specificity than previously identified, oxidizing both mercaptopropionate and mercaptopyruvate thiol and persulfide substrates. The results obtained from these investigations provide insight into the overall mechanism and physiological role of the mdo operon in sulfide oxidation and assimilation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp-protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

"Allosteric" was first introduced to mean the other site (i.e., a site distinct from the active or orthosteric site), an adjective for "regulation" to imply a regulatory outcome resulting from ligand binding at another site. That original idea outlines a system with two ligand-binding events at two distinct locations on a macromolecule (originally a protein system), which defines a four-state energy cycle. An allosteric energy cycle provides a quantifiable allosteric coupling constant and focuses our attention on the unique properties of the four equilibrated protein complexes that constitute the energy cycle. Because many observed phenomena have been referenced as "allosteric regulation" in the literature, the goal of this work is to use literature examples to explore which systems are and are not consistent with the two-ligand thermodynamic energy cycle-based definition of allosteric regulation. We emphasize the need for consistent language so comparisons can be made among the ever-increasing number of allosteric systems. Building on the mutually exclusive natures of an energy cycle definition of allosteric regulation versus classic two-state models, we conclude our discussion by outlining how the often-proposed Rube-Goldberg-like mechanisms are likely inconsistent with an energy cycle definition of allosteric regulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeting mitochondrial oxidative phosphorylation (OXPHOS) to treat cancer has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Herein, comprehensive mitochondrial phenotyping was leveraged to define both the composition and function of OXPHOS across various murine cancers and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were discovered to be consistently lower across distinct cancer types. Assuming intrinsic OXPHOS expression/function predicts OXPHOS reliance in vivo, these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across cancer types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given cancer type., (© 2023. Springer Nature Limited.)

The FMN reductases (SsuE and MsuE of the alkanesulfonate monooxygenase systems) supply reduced flavin to their partner monooxygenases for the desulfonation of alkanesulfonates. Flavin reductases that comprise two-component systems must be able to regulate both flavin reduction and transfer. One mechanism to control these distinct processes is through changes in the oligomeric state of the enzymes. Despite their similar overall structures, SsuE and MsuE showed clear differences in their oligomeric states in the presence of substrates. The oligomeric state of SsuE was converted from a tetramer to a dimer/tetramer equilibrium in the presence of FMN or NADPH in analytical ultracentrifugation studies. Conversely, MsuE shifted from a dimer to a single tetrameric state with FMN, and the NADPH substrate did not induce a similar oligomeric shift. There was a fast tetramer to dimer equilibrium shift occurring at the dimer/dimer interface in H/D-X investigations with apo SsuE. Formation of the SsuE/FMN complex slowed the tetramer/dimer conversion, leading to a slower exchange along the dimer/dimer interface. The oligomeric shift of the MsuE/FMN complex from a dimer to a distinct tetramer showed a decrease in H/D-X in the region around the π-helices at the dimer/dimer interface. Both SsuE and MsuE showed a comparable and significant increase in the melting temperature with the addition of FMN, indicating the conformers formed by each FMN-bound enzyme had increased stability. A mechanism that supports the different structural shifts is rationalized by the different roles these enzymes play in providing reduced flavin to single or multiple monooxygenase enzymes.

METTL16, a human m6A RNA methyltransferase, is currently known for its modification of U6 and MAT2A RNAs. Several studies have identified additional RNAs to which METTL16 binds, however whether METTL16 modifies these RNAs is still in question. Moreover, a recent study determined that METTL16 contains more than one RNA-binding domain, leaving the importance of each individual RNA-binding domain unknown. Here we examined the effects of mutating the METTL16 protein in certain domains on overall cell processes. We chose to mutate the N-terminal RNA-binding domain, the methyltransferase domain, and the C-terminal RNA-binding domain. With these mutants, we identified changes in RNA-binding ability, protein and RNA expression, cell cycle phase occupancy, and proliferation. From the resulting changes in RNA and protein expression, we saw effects on cell cycle, metabolism, intracellular transport, and RNA processing pathways, which varied between the METTL16 mutant lines. We also saw significant effects on the G1 and S phase occupancy times and proliferative ability with some but not all the mutants. We have therefore concluded that while METTL16 may or may not m6A-modify all RNAs it binds, its binding (or lack of) has a significant outcome on a variety of cell processes.

Multidrug-resistant bacteria cause immense public health concerns as once effective antibiotics no longer work against even common infections. Concomitantly, there has been a decline in the discovery of new antibiotics, and the current global clinical pipeline is woefully inadequate, especially against resistant Gram-negative bacteria. One major contribution to Gram-negative resistance is the presence of a protective outer membrane. Consequently, an appealing option for tackling resistance is to adversely affect that outer membrane. With that in mind, we define the response regulator PhoP as a target for new 2-aminoimidazole compounds and show that they affect the integrity of the outer membrane in resistant strains of Escherichia coli and Klebsiella pneumoniae . We also provide empirical evidence for the 2-aminoimidazole mechanism of action.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5') pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McLaughlin, Nelson, Coalson, Hagen, Montgomery, Wooten, Zeczycki, Vohra and Fisher-Wellman.)

Opioids are not universally effective for treating neuropathic pain following spinal cord injury (SCI), a finding that we previously demonstrated in a rat model of SCI. The aim of this study was to determine analgesic response of morphine-responsive and nonresponsive SCI rats to adjunct treatment with dopamine modulators and to establish if the animal groups expressed distinct metabolomic profiles. Thermal thresholds were tested in female Long Evans rats (N = 45) prior to contusion SCI, after SCI and following injection of morphine, morphine combined with dopamine modulators, or dopamine modulators alone. Spinal cord and striatum samples were processed for metabolomics and targeted mass spectrometry. Morphine provided analgesia in 1 of 3 of SCI animals. All animals showed improved analgesia with morphine + pramipexole (D3 receptor agonist). Only morphine nonresponsive animals showed improved analgesia with the addition of SCH 39166 (D1 receptor antagonist). Metabolomic analysis identified 3 distinct clusters related to the tyrosine pathway that corresponded to uninjured, SCI morphine-responsive and SCI morphine-nonresponsive groups. Mass spectrometry showed matching differences in dopamine levels in striatum and spinal cord between these groups. The data suggest an overall benefit of the D3 receptor system in improving analgesia, and an association between morphine responsiveness and metabolomic changes in the tyrosine/dopamine pathways in striatum and spinal cord. PERSPECTIVE: Spinal cord injury (SCI) leads to opioid-resistant neuropathic pain that is associated with changes in dopamine metabolomics in the spinal cord and striatum of rats. We present evidence that adjuvant targeting of the dopamine system may be a novel pain treatment approach to overcome opioid desensitization and tolerance after SCI., (Copyright © 2021 United States Association for the Study of Pain, Inc. All rights reserved.)

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease (PAD) and is characterized by high rates of morbidity and mortality. As with most severe cardiovascular disease manifestations, Black individuals disproportionately present with CLI. Accordingly, there remains a clear need to better understand the reasons for this discrepancy and to facilitate personalized therapeutic options specific for this population. Gastrocnemius muscle was obtained from White and Black healthy adult volunteers and patients with CLI for whole transcriptome shotgun sequencing (WTSS) and enrichment analysis was performed to identify alterations in specific Reactome pathways. When compared to their race-matched healthy controls, both White and Black patients with CLI demonstrated similar reductions in nuclear and mitochondrial encoded genes and mitochondrial oxygen consumption across multiple substrates, indicating a common bioenergetic paradigm associated with amputation outcomes regardless of race. Direct comparisons between tissues of White and Black patients with CLI revealed hemostasis, extracellular matrix organization, platelet regulation, and vascular wall interactions to be uniquely altered in limb muscles of Black individuals. Among traditional vascular growth factor signaling targets, WTSS revealed only Tie1 to be significantly altered from White levels in Black limb muscle tissues. Quantitative reverse transcription polymerase chain reaction validation of select identified targets verified WTSS directional changes and supports reductions in MMP9 and increases in NUDT4P1 and GRIK2 as unique to limb muscles of Black patients with CLI. This represents a critical first step in better understanding the transcriptional program similarities and differences between Black and White patients in the setting of amputations related to CLI and provides a promising start for therapeutic development in this population.

Exercise capacity is a strong predictor of all-cause mortality. Skeletal muscle mitochondrial respiratory capacity, its biggest contributor, adapts robustly to changes in energy demands induced by contractile activity. While transcriptional regulation of mitochondrial enzymes has been extensively studied, there is limited information on how mitochondrial membrane lipids are regulated. Here, we show that exercise training or muscle disuse alters mitochondrial membrane phospholipids including phosphatidylethanolamine (PE). Addition of PE promoted, whereas removal of PE diminished, mitochondrial respiratory capacity. Unexpectedly, skeletal muscle-specific inhibition of mitochondria-autonomous synthesis of PE caused respiratory failure because of metabolic insults in the diaphragm muscle. While mitochondrial PE deficiency coincided with increased oxidative stress, neutralization of the latter did not rescue lethality. These findings highlight the previously underappreciated role of mitochondrial membrane phospholipids in dynamically controlling skeletal muscle energetics and function.

Pyruvate kinase plays a central role in glucose catabolism in bacteria, and efficient utilization of this hexose has been linked to the virulence of Brucella strains in mice. The brucellae produce a single pyruvate kinase which is an ortholog of the Bradyrhizobium manganese (Mn)-dependent pyruvate kinase PykM. A biochemical analysis of the Brucella pyruvate kinase and phenotypic analysis of a Brucella abortus mutant defective in high-affinity Mn import indicate that this enzyme is an authentic PykM ortholog which functions as a Mn-dependent enzyme in vivo The loss of PykM has a negative impact on the capacity of the parental 2308 strain to utilize glucose, fructose, and galactose but not on its ability to utilize ribose, xylose, arabinose, or erythritol, and a pykM mutant displays significant attenuation in C57BL/6 mice. Although the enzyme pyruvate phosphate dikinase (PpdK) can substitute for the loss of pyruvate kinase in some bacteria and is also an important virulence determinant in Brucella , a phenotypic analysis of B. abortus 2308 and isogenic pykM , ppdK , and pykM ppdK mutants indicates that PykM and PpdK make distinctly different contributions to carbon metabolism and virulence in these bacteria. IMPORTANCE Mn plays a critical role in the physiology and virulence of Brucella strains, and the results presented here suggest that one of the important roles that the high-affinity Mn importer MntH plays in the pathogenesis of these strains is supporting the function of the Mn-dependent kinase PykM. A better understanding of how the brucellae adapt their physiology and metabolism to sustain their intracellular persistence in host macrophages will provide knowledge that can be used to design improved strategies for preventing and treating brucellosis, a disease that has a significant impact on both the veterinary and public health communities worldwide., (Copyright © 2018 American Society for Microbiology.)

The most severe manifestation of peripheral arterial disease (PAD) is critical limb ischemia (CLI). CLI patients suffer high rates of amputation and mortality; accordingly, there remains a clear need both to better understand CLI and to develop more effective treatments. Gastrocnemius muscle was obtained from 32 older (51-84 years) non-PAD controls, 27 claudicating PAD patients (ankle-brachial index [ABI] 0.65 ± 0.21 SD), and 19 CLI patients (ABI 0.35 ± 0.30 SD) for whole transcriptome sequencing and comprehensive mitochondrial phenotyping. Comparable permeabilized myofiber mitochondrial function was paralleled by both similar mitochondrial content and related mRNA expression profiles in non-PAD control and claudicating patient tissues. Tissues from CLI patients, despite being histologically intact and harboring equivalent mitochondrial content, presented a unique bioenergetic signature. This signature was defined by deficits in permeabilized myofiber mitochondrial function and a unique pattern of both nuclear and mitochondrial encoded gene suppression. Moreover, isolated muscle progenitor cells retained both mitochondrial functional deficits and gene suppression observed in the tissue. These findings indicate that muscle tissues from claudicating patients and non-PAD controls were similar in both their bioenergetics profile and mitochondrial phenotypes. In contrast, CLI patient limb skeletal muscles harbor a unique skeletal muscle mitochondriopathy that represents a potentially novel therapeutic site for intervention.

Naturally or surgically induced postmenopausal women are widely prescribed estrogen therapies to alleviate symptoms associated with estrogen loss and to lower the subsequent risk of developing metabolic diseases, including diabetes and nonalcoholic fatty liver disease. However, the molecular mechanisms by which estrogens modulate metabolism across tissues remain ill-defined. We have previously reported that 17β-estradiol (E2) exerts antidiabetogenic effects in ovariectomized (OVX) mice by protecting mitochondrial and cellular redox function in skeletal muscle. The liver is another key tissue for glucose homeostasis and a target of E2 therapy. Thus, in the present study we determined the effects of acute loss of ovarian E2 and E2 administration on liver mitochondria. In contrast to skeletal muscle mitochondria, E2 depletion via OVX did not alter liver mitochondrial respiratory function or complex I (CI) specific activities (NADH oxidation, quinone reduction, and H 2 O 2 production). Surprisingly, in vivo E2 replacement therapy and in vitro E2 exposure induced tissue-specific effects on both CI activity and on the rate and topology of CI H 2 O 2 production. Overall, E2 therapy protected and restored the OVX-induced reduction in CI activity in skeletal muscle, whereas in liver mitochondria E2 increased CI H 2 O 2 production and decreased ADP-stimulated respiratory capacity. These results offer novel insights into the tissue-specific effects of E2 on mitochondrial function., (© 2018 Torres et al.)

Cardiolipin (CL) is an anionic phospholipid mainly located in the inner mitochondrial membrane, where it helps regulate bioenergetics, membrane structure, and apoptosis. Localized, phase-segregated domains of CL are hypothesized to control mitochondrial inner membrane organization. However, the existence and underlying mechanisms regulating these mitochondrial domains are unclear. Here, we first isolated detergent-resistant cardiac mitochondrial membranes that have been reported to be CL-enriched domains. Experiments with different detergents yielded only nonspecific solubilization of mitochondrial phospholipids, suggesting that CL domains are not recoverable with detergents. Next, domain formation was investigated in biomimetic giant unilamellar vesicles (GUVs) and newly synthesized giant mitochondrial vesicles (GMVs) from mouse hearts. Confocal fluorescent imaging revealed that introduction of cytochrome c into membranes promotes macroscopic proteolipid domain formation associated with membrane morphological changes in both GUVs and GMVs. Domain organization was also investigated after lowering tetralinoleoyl-CL concentration and substitution with monolyso-CL, two common modifications observed in cardiac pathologies. Loss of tetralinoleoyl-CL decreased proteolipid domain formation in GUVs, because of a favorable Gibbs-free energy of lipid mixing, whereas addition of monolyso-CL had no effect on lipid mixing. Moreover, murine GMVs generated from cardiac acyl-CoA synthetase-1 knockouts, which have remodeled CL acyl chains, did not perturb proteolipid domains. Finally, lowering the tetralinoleoyl-CL content had a stronger influence on the oxidation status of cytochrome c than did incorporation of monolyso-CL. These results indicate that proteolipid domain formation in the cardiac mitochondrial inner membrane depends on tetralinoleoyl-CL concentration, driven by underlying lipid-mixing properties, but not the presence of monolyso-CL., (© 2018 Pennington et al.)

The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM-integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these "dead" enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.

The Ca 2+ -dependent deamidation and transamidation activities of transglutaminase 2 (TG2) are important to numerous physiological and pathological processes. Herein, we have examined the steady-state kinetics and 15 (V/K) kinetic isotope effects (KIEs) for the TG2-catalyzed deamidation and transamidation of N-Benzyloxycarbonyl-l-Glutaminylglycine (Z-Gln-Gly) using putrescine as the acyl acceptor substrate. Kinetic parameters determined from initial velocity plots are consistent with previously proposed mechanisms. Significant differences in the 15 (V/K) KIEs on NH 3 release determined for the deamidation (0.2%) and the transamidation (2.3%) of Z-Gln-Gly suggest the rate-limiting steps of TG2 active site acylation are dependent on the presence of the acyl acceptor. We propose a plausible mechanistic explanation where substrate-induced conformational changes may play a role in promoting catalysis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cardiac mitochondrial phospholipid acyl chains regulate respiratory enzymatic activity. In several diseases, the rodent cardiac phospholipidome is extensively rearranged; however, whether specific acyl chains impair respiratory enzyme function is unknown. One unique remodeling event in the myocardium of obese and diabetic rodents is an increase in docosahexaenoic acid (DHA) levels. Here, we first confirmed that cardiac DHA levels are elevated in diabetic humans relative to controls. We then used dietary supplementation of a Western diet with DHA as a tool to promote cardiac acyl chain remodeling and to study its influence on respiratory enzyme function. DHA extensively remodeled the acyl chains of cardiolipin (CL), mono-lyso CL, phosphatidylcholine, and phosphatidylethanolamine. Moreover, DHA lowered enzyme activities of respiratory complexes I, IV, V, and I+III. Mechanistically, the reduction in enzymatic activities were not driven by a dramatic reduction in the abundance of supercomplexes. Instead, replacement of tetralinoleoyl-CL with tetradocosahexaenoyl-CL in biomimetic membranes prevented formation of phospholipid domains that regulate enzyme activity. Tetradocosahexaenoyl-CL inhibited domain organization due to favorable Gibbs free energy of phospholipid mixing. Furthermore, in vitro substitution of tetralinoleoyl-CL with tetradocosahexaenoyl-CL blocked complex-IV binding. Finally, reintroduction of linoleic acid, via fusion of phospholipid vesicles to mitochondria isolated from DHA-fed mice, rescued the major losses in the mitochondrial phospholipidome and complexes I, IV, and V activities. Altogether, our results show that replacing linoleic acid with DHA lowers select cardiac enzyme activities by potentially targeting domain organization and phospholipid-protein binding, which has implications for the ongoing debate about polyunsaturated fatty acids and cardiac health., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Menopause results in a progressive decline in 17β-estradiol (E2) levels, increased adiposity, decreased insulin sensitivity, and a higher risk for type 2 diabetes. Estrogen therapies can help reverse these effects, but the mechanism(s) by which E2 modulates susceptibility to metabolic disease is not well understood. In young C57BL/6N mice, short-term ovariectomy decreased-whereas E2 therapy restored-mitochondrial respiratory function, cellular redox state (GSH/GSSG), and insulin sensitivity in skeletal muscle. E2 was detected by liquid chromatography-mass spectrometry in mitochondrial membranes and varied according to whole-body E2 status independently of ERα. Loss of E2 increased mitochondrial membrane microviscosity and H 2 O 2 emitting potential, whereas E2 administration in vivo and in vitro restored membrane E2 content, microviscosity, complex I and I + III activities, H 2 O 2 emitting potential, and submaximal OXPHOS responsiveness. These findings demonstrate that E2 directly modulates membrane biophysical properties and bioenergetic function in mitochondria, offering a direct mechanism by which E2 status broadly influences energy homeostasis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer.

Cardiac phospholipids, notably cardiolipin, undergo acyl chain remodeling and/or loss of content in aging and cardiovascular diseases, which is postulated to mechanistically impair mitochondrial function. Less is known about how diet-induced obesity influences cardiac phospholipid acyl chain composition and thus mitochondrial responses. Here we first tested if a high fat diet remodeled murine cardiac mitochondrial phospholipid acyl chain composition and consequently disrupted membrane packing, supercomplex formation and respiratory enzyme activity. Mass spectrometry analyses revealed that mice consuming a high fat diet displayed 0.8-3.3 fold changes in cardiac acyl chain remodeling of cardiolipin, phosphatidylcholine, and phosphatidylethanolamine. Biophysical analysis of monolayers constructed from mitochondrial phospholipids of obese mice showed impairment in the packing properties of the membrane compared to lean mice. However, the high fat diet, relative to the lean controls, had no influence on cardiac mitochondrial supercomplex formation, respiratory enzyme activity, and even respiration. To determine if the effects were tissue specific, we subsequently conducted select studies with liver tissue. Compared to the control diet, the high fat diet remodeled liver mitochondrial phospholipid acyl chain composition by 0.6-5.3-fold with notable increases in n-6 and n-3 polyunsaturation. The remodeling in the liver was accompanied by diminished complex I to III respiratory enzyme activity by 3.5-fold. Finally, qRT-PCR analyses demonstrated an upregulation of liver mRNA levels of tafazzin, which contributes to cardiolipin remodeling. Altogether, these results demonstrate that diet-induced obesity remodels acyl chains in the mitochondrial phospholipidome and exerts tissue specific impairments of respiratory enzyme activity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The objective of our study was to determine the mechanism of action of the short-chain ceramide analog, C6-ceramide, and the breast cancer drug, tamoxifen, which we show coactively depress viability and induce apoptosis in human acute myelogenous leukemia cells. Exposure to the C6-ceramide-tamoxifen combination elicited decreases in mitochondrial membrane potential and complex I respiration, increases in reactive oxygen species (ROS), and release of mitochondrial proapoptotic proteins. Decreases in ATP levels, reduced glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins also resulted. Cytotoxicity of the drug combination was mitigated by exposure to antioxidant. Cells metabolized C6-ceramide by glycosylation and hydrolysis, the latter leading to increases in long-chain ceramides. Tamoxifen potently blocked glycosylation of C6-ceramide and long-chain ceramides. N-desmethyltamoxifen, a poor antiestrogen and the major tamoxifen metabolite in humans, was also effective with C6-ceramide, indicating that traditional antiestrogen pathways are not involved in cellular responses. We conclude that cell death is driven by mitochondrial targeting and ROS generation and that tamoxifen enhances the ceramide effect by blocking its metabolism. As depletion of ATP and targeting the "Warburg effect" represent dynamic metabolic insult, this ceramide-containing combination may be of utility in the treatment of leukemia and other cancers., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

Protein structure, ligand binding, and catalytic turnover contributes to the governance of catalytic events occurring at spatially distinct domains in multifunctional enzymes. Coordination of these catalytic events partially rests on the ability of spatially discrete active sites to communicate with other allosteric and active sites on the same polypeptide chain (intramolecular) or on different polypeptide chains (intermolecular) within the holoenzyme. Often, communication results in long-range effects on substrate binding or product release. For example, pyruvate binding to the carboxyl transferase (CT) domain of pyruvate carboxylase (PC) increases the rate of product release in the biotin carboxylase (BC) domain. In order to address how CT domain ligand occupancy is "sensed" by other domains, we generated functional, mixed hybrid tetramers using the E218A (inactive BC domain) and T882S (low pyruvate binding, low activity) mutant forms of PC. The apparent Ka pyruvate for the pyruvate-stimulated release of Pi catalyzed by the T882S:E218A[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that for the T882S homotetramer. In addition, the ratio of the rates of oxaloacetate formation to Pi release for the WT:T882S[1:1] and E218A:T882S[1:1] hybrid tetramer-catalyzed reactions was 0.5 and 0.6, respectively, while the T882S homotetramer exhibited a near 1:1 coupling of the two domains, suggesting that the mechanisms coordinating catalytic events is more complicated that we initially assumed. The results presented here are consistent with an intermolecular communication mechanism, where pyruvate binding to the CT domain is "sensed" by domains on a different polypeptide chain within the tetramer.

Activities of the enzymes involved in cellular respiration are markedly influenced by the composition of the phospholipid environment of the inner mitochondrial membrane. Contrary to previous suppositions, we show that fusion of mitochondria isolated from healthy cardiac muscle with cardiolipin or dioleoylphosphatidylcholine results in a 2-6-fold reduction in the activity of complexes I, II, and IV. The activity of complex III was unaffected by increased phospholipid levels. Phospholipid content had an indiscriminate yet detrimental effect on the combined activities of complexes I+III and II+III. These results have strong implications for therapeutic lipid replacement strategies, in which phospholipid modification of the mitochondria is proposed to enhance mitochondrial function.

It is essential to understand the role of cardiolipin (CL) in mitochondrial membrane organization given that changes in CL levels contribute to mitochondrial dysfunction in type II diabetes, ischemia-reperfusion injury, heart failure, breast cancer, and aging. Specifically, there are contradictory data on how CL influences the molecular packing of membrane phospholipids. Therefore, we determined how increasing levels of heart CL impacted molecular packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures found within the mitochondrial inner membrane, using merocyanine (MC540) fluorescence. We broadly categorized lipid vesicles of equal mass as loosely packed, intermediate, and highly packed based on peak MC540 fluorescence intensity. CL had opposite effects on loosely versus highly packed vesicles. Exposure of loosely packed vesicles to increasing levels of CL dose-dependently increased membrane packing. In contrast, increasing amounts of CL in highly packed vesicles decreased the packing in a dose-dependent manner. In vesicles that were categorized as intermediate packing, CL had either no effect or decreased packing at select doses in a dose-independent manner. Altogether, the results aid in resolving some of the discrepant data by demonstrating that CL displays differential effects on membrane packing depending on the composition of the lipid environment. This has implications for mitochondrial protein activity in response to changing CL levels in microdomains of varying composition., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Numerous steady-state kinetic studies have examined the complex catalytic reaction mechanism of the multifunctional enzyme, pyruvate carboxylase (PC). Through initial velocity, product inhibition, isotopic exchange and alternate substrate experiments, early investigators established that PC catalyzes the MgATP-dependent carboxylation of pyruvate by HCO3 (-) through a nonclassical sequential Bi Bi Uni Uni reaction mechanism. This review surveys previous steady-state kinetic investigations of PC and evaluates the proposed hypotheses concerning the overall catalytic mechanism, nonlinear kinetics and active site coupling in the context of recent structural and mutagenic analyses of this multifunctional enzyme. The determination several PC holoenzyme structures have aided in corroborating the proposed molecular mechanisms by which catalysis occurs and established the inextricable link between the dynamic protein motions and complex kinetic mechanisms associated with PC activity. Unexpectedly, the conclusions drawn from these early steady-state kinetic investigations have consistently proven to be in fundamental agreement with our current understanding of PC catalysis, which is a testament to the overarching sophistication of the methods pioneered by Michaelis and Menten and further developed by Northrop, Cleland and others., (© 2014 FEBS.)

Oxamate, an isosteric and isoelectronic inhibitory analogue of pyruvate, enhances the rate of enzymatic decarboxylation of oxaloacetate in the carboxyl transferase domain of pyruvate carboxylase (PC). It is unclear, though, how oxamate exerts a stimulatory effect on the enzymatic reaction. Herein, we report direct (13)C nuclear magnetic resonance (NMR) evidence that oxamate acts as a carboxyl acceptor, forming a carbamylated oxamate product and thereby accelerating the enzymatic decarboxylation reaction. (13)C NMR was used to monitor the PC-catalyzed formation of [4-(13)C]oxaloacetate and subsequent transfer of (13)CO(2) from oxaloacetate to oxamate. In the presence of oxamate, the apparent K(m) for oxaloacetate is artificially suppressed (from 15 to 4-5 μM). Interestingly, the steady-state kinetic analysis of the initial rates determined at varying concentrations of oxaloacetate and fixed concentrations of oxamate revealed initial velocity patterns inconsistent with a simple ping-pong-type mechanism. Rather, the patterns suggest the existence of an alternate decarboxylation pathway in which an unstable intermediate is formed. The steady-state kinetic analysis coupled with the normal (13)(V/K) kinetic isotope effect observed on C-4 of oxaloacetate [(13)(V/K) = 1.0117 ± 0.0005] indicates that the transfer of CO(2) from carboxybiotin to oxamate is the partially rate-limiting step of the enzymatic reaction. The catalytic mechanism proposed for the carboxylation of oxamate is similar to that proposed for the carboxylation of pyruvate, which occurs via the formation of an enol intermediate.

Mutation of Arg427 and Arg472 in Rhizobium etli pyruvate carboxylase to serine or lysine greatly increased the activation constant (K(a)) of acetyl CoA, with the increase being greater for the Arg472 mutants. These results indicate that while both these residues are involved in the binding of acetyl CoA to the enzyme, Arg472 is more important than Arg427. The mutations had substantially smaller effects on the k(cat) for pyruvate carboxylation. Part of the effects of the mutations was to increase the K(m) for MgATP and the K(a) for activation by free Mg(2+) determined at saturating acetyl CoA concentrations. The inhibitory effects of the mutations on the rates of the enzyme-catalyzed bicarbonate-dependent ATP cleavage, carboxylation of biotin, and phosphorylation of ADP by carbamoyl phosphate indicate that the major locus of the effects of the mutations was in the biotin carboxylase (BC) domain active site. Even though both Arg427 and Arg472 are distant from the BC domain active site, it is proposed that their contacts with other residues in the allosteric domain, either directly or through acetyl CoA, affect the positioning and orientation of the biotin-carboxyl carrier protein (BCCP) domain and thus the binding of biotin at the BC domain active site. On the basis of the kinetic analysis proposed here, it is proposed that mutations of Arg427 and Arg472 perturb these contacts and consequently the binding of biotin at the BC domain active site. Inhibition of pyruvate carboxylation by the allosteric inhibitor l-aspartate was largely unaffected by the mutation of either Arg427 or Arg472.

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.

In this review we examine the effects of the allosteric activator, acetyl CoA on both the structure and catalytic activities of pyruvate carboxylase. We describe how the binding of acetyl CoA produces gross changes to the quaternary and tertiary structures of the enzyme that are visible in the electron microscope. These changes serve to stabilize the tetrameric structure of the enzyme. The main locus of activation of the enzyme by acetyl CoA is the biotin carboxylation domain of the enzyme where ATP-cleavage and carboxylation of the biotin prosthetic group occur. As well as enhancing reaction rates, acetyl CoA also enhances the binding of some substrates, especially HCO3-, and there is also a complex interaction with the binding of the cofactor Mg2. The activation of pyruvate carboxylase by acetyl CoA is generally a cooperative processes, although there is a large degree of variability in the degree of cooperativity exhibited by the enzyme from different organisms. The X-ray crystallographic holoenzyme structures of pyruvate carboxylases from Rhizobium etli and Staphylococcus aureus have shown the allosteric acetyl CoA binding domain to be located at the interfaces of the biotin carboxylation and carboxyl transfer and the carboxyl transfer and biotin carboxyl carrier protein domains., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family.

The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO(3)(-) deprotonation (Glu305 and Arg301), and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO(3)(-), Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5- and 4-fold increase in k(cat) for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO(3)(-) by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO(2) and PO(4)(3-). PO(4)(3-) then acts as the base to deprotonate the tethered biotin at the N(1)-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO(2) to give carboxybiotin. The formation of a distinct salt bridge between Arg353 and Glu248 is proposed to aid in partially precluding carboxybiotin from reentering the biotin carboxylase active site, thus preventing its premature decarboxylation prior to the binding of a carboxyl acceptor in the carboxyl transferase domain.

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described.

2',3'-O-(2,4,6-Trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme. At concentrations above saturating, MgATP activates bicarbonate-dependent ATP cleavage, but inhibits the overall reaction. The fluorescence of MgTNP-ATP increases by about 2.5-fold upon binding to the enzyme and decreases on addition of saturating acetyl CoA. However, not all the MgTNP-ATP is displaced by acetyl CoA, or with a combination of saturating concentrations of MgATP and acetyl CoA. The kinetics of the binding of MgTNP-ATP to pyruvate carboxylase have been measured and shown to be triphasic, with the two fastest phases having pseudo first-order rate constants that are dependent on the concentration of MgTNP-ATP. The kinetics of displacement from the enzyme by acetyl CoA have been measured and also shown to be triphasic. A model of the binding process is proposed that links the kinetics of MgTNP-ATP binding to the allosteric activation of the enzyme., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilization of the quaternary structure of the enzyme, suggesting that this residue has a structural role. Mutations R548K, Q552N, and Q552A resulted in a loss of the ability to catalyze pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate, and the exchange of protons between pyruvate and water. These mutants retained the ability to catalyze reactions that occur at the active site of the biotin carboxylase domain, i.e., bicarbonate-dependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalyzed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and the subsequent transfer of protons between pyruvate and biotin in the partial reaction catalyzed in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase.

This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin - biotin, FTP - ATP binding site, oxamate - pyruvate binding site, phosphonoacetate - binding site of the putative carboxyphosphate intermediate.

The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate, and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain but surprisingly showed 7- and 3.5-fold increases in activity, as compared to that of the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A-catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and removal of a proton from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO(2) to form oxaloacetate and complete the reaction.

The tetrametallic ruthenium-oxo-hydroxo-hydride complex {[(PCy(3))(CO)RuH](4)(mu(4)-O)(mu(3)-OH)(mu(2)-OH)} (1) was synthesized in two steps from the monomeric complex (PCy(3))(CO)RuHCl (2). The tetrameric complex 1 was found to be a highly effective catalyst for the transfer dehydrogenation of alcohols. Complex 1 showed a different catalytic activity pattern towards primary and secondary benzyl alcohols, as indicated by the Hammett correlation for the oxidation reaction of p-X-C(6)H(4)CH(2)OH (rho = -0.45) and p-X-C(6)H(4)CH(OH)CH(3) (rho = +0.22) (X = OMe, CH(3), H, Cl, CF(3)). Both a sigmoidal curve from the plot of initial rate vs [PhCH(OH)CH(3)] (K(0.5) = 0.34 M; Hill coefficient, n = 4.2+/-0.1) and the phosphine inhibition kinetics revealed the highly cooperative nature of the complex for the oxidation of secondary alcohols.