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Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction. acyl-enzyme | enzyme mechanism | steady state | reaction trajectory

A classical peptide inhibitor of serine proteases that is hydrolyzed [approximately equal to] [10.sup.7] times more slowly than a good substrate is shown to form an acyl-enzyme intermediate rapidly. Despite this quick first step, further reaction is slowed dramatically because of tight and oriented binding of the cleaved peptide, preventing acyl-enzyme hydrolysis and favoring the reverse reaction. Moreover, this mechanism appears to be common to a large class of tight-binding serine protease inhibitors that mimic good substrates. The arrest of enzymatic reaction at the intermediate stage allowed us to determine that the consensus nucleophilic attack angle is close to 90 in the reactive Michaelis complexes.

Crystallographic studies demonstrate that the S113E complex, containing isocitrate dehydrogenase, isopropylmalate, NADP, and magnesium ions, exhibits alterations in global and active site structures. the altered complex together with host of other related changes cause switching of the substrate specificity.

Research describes engineering the structure of isocitrate dehydrogenase by a combination of rational and random mutagenesis to alter the enzyme's substrate preference to isopropylmalate from isocitrate. While substitution reactions involving amino acid residues at position 113, 115, and 116 bring about the switch, asparagine115 seems to be more specifically involved.

The structural changes in the aspartate receptor of Escherichia coli were determined through the incorporation of cysteine residues in the cytoplasmic domain at position 265 and in the periplasmic domain at position 36. The aspartate receptor is present in the bacterial inner membrane and is considered as an homologous transmembrane receptor influencing chemotactic reactions. It was discovered that the aspartate receptor exhibited conformational changes during covalent methylation for each of the four methylation sites found in its cytoplasmic domain.

Spin-labeling electron paramagnetic resonance (EPR) was utilized to examine conformational changes in the Salmonella typhimurium aspartate receptor (AR) induced by ligand-binding. The EPR spectra of labeled proteins were determined in the presence and absence of the ligand aspartate while site-directed mutagenesis was used to mutate the gene encoding the wild-type AR to generate cysteine-containing mutant AR. Results showed that the aspartate did not change the spectra of AR spin labels.

Cocrystallization of isocitrate dehydrogenase and isocitrate dehydrogenase kinase/phosphatase was attempted in the presence of an ATP analog, to further clarify phosphorylation regulation of isocitrate dehydrogenase. Cocrystallization was unsuccessful, but a new crystal form of isocitrate dehydrogenase was obtained, related to a previously reported tetragonal form.

A study was conducted on the mechanisms of methylation of the Escherichia coli chemotaxis receptors. The findings indicate that C-terminally truncated aspartate receptors impaired in methyltransferase binding are efficiently methylated when present in membranes. Moreover, the rates of interdimer methylation of the truncated receptors were lower than that of the unmodified aspartate receptor.

The targeted disulfide cross-linking procedure was used on the cytoplasmic domain of the aspartate receptor of Salmonella typhimurium. Results revealed a generally alpha-helical conformation of the linker region, and a close juxtaposition and a parallel alignment at the interface between the two subunits in the linker region. Aspartate binding in the periplasmic domain induces a closer juxtaposition of the two subunits in the cytoplasmic domain.

The synthesis and photochemical characterization of two caged NADP compounds as well as the static X-ray structures of the compounds were described through utilization of laser photolysis. Results have shown that one compound can bind to isocitrate dehydrogenase (IDH) prior to photolysis but is not catalytically active. Another compound can inhibit binding of the IDH prior to photolysis.

Site-directed mutagenesis of the aspartate receptor of Salmonella typhimurium (TarS) at serine 68, a residue situated within the aspartate binding pocket and at the subunit interface, identified this residue as an allosteric switch in this receptor. Negative cooperativity was found in the wild-type receptor and was maintained by the mutations S68C and S68D. No cooperativity was found in the mutant S68A. Positive cooperativity was shown by the substitutions S68T, S68L, S68N and S68I.

Photolabile derivatives of isocitrate were synthesized, characterized and subjected to photolysis to determine conversion intermediates and chemical characteristics. X-ray crystal structure studies that involved subjecting photolabile isocitrate mixed with Mg2+in the absence of nicotinamide adenine dinucleotide phosphate to photolysis detected the photolysed enzyme containing Mg2+ at many of the metal binding sites and the liberated isocitrate bound into an enzyme complex.

Introduction of mutant aspartate receptors with methylation site substitutions into cells and assays for chemotactic ability help study the influence of multiple site methylation on chemotactic behavior. Mutation of methylation sites impairs chemotaxis, with the effect being more severe for a rapidly methylated site. Chemotaxis in the wild-type strain is only slightly influenced by the expression of mutant receptors. In vivo assays reveal that the lowered rates of methylation increase due to aspartate cause defects in chemotaxis in most of the mutants.

A study involved the measurement of the kinematic values of attractant binding of the ligand region of chemotaxis aspartate receptor. The structural regions involved in attractant-induced conformational change were located. Each subunit of the homodimer revealed six parafluorophenylalanine (4-F-Phe) positions. The dissociation rate constant (k-off approximately equal to 10 cubed per second) was calculated from the association rate constant (K-on approximately equal to 10 to the 9th power meter per second) of the bond between the isolated periplasmic domain and the aspartate.

An analysis of structural, chemical and cooperative properties of Escherichia coli and Salmonella typhimurium aspartate receptors reveals that both have specific negatively cooperative binding configurations. Amino acid sequences for aspartate receptors have 79% identity but the E. coli receptor exhibits single site binding. Consequently, the E. coli receptor binds with 'half-of-the sites' cooperativity, where the E. coli receptor's second ligand does not bind.

The isocitrate dehydrogenase of Escherichia coli exhibits a kinetic mechanism, which can be understood by studying product inhibitors, dead-end inhibitors, optional substrates, and site-directed mutant of the enzyme. Steady-state disordered mechanisms are observed in the enzymes, despite the lack of similarities in the basic series and the lack of the Rossmann fold in the IDH Escherichia coli enzyme. Kinetic mechanisms exhibit a convergent progress, resulting in varying geometries which are employed to recognize the characteristics of the geometry of the coenzyme.

Alpha-ketoglutarates exhibit the use of isocitrate dehydrogenase (IDH), whose conformation is analyzed by examining the geometry of IDH complexed with alpha-ketoglutarate, NADPH, and Ca2+. Isocitrate or alpha-ketoglutarate, in association with calcium, binds to the enzyme as a complex, thereby totally preventing turnover. A section of enzyme catalysts are observed in isocitrate dehydrogenase, revealing a template-type system with a substrate which stimulates some conformational alterations in the forward reaction.

A bound ternary complex of isocitrate, NADP+ and Ca2+, is used to determine the geometry of isocitrate dehydrogenase (IDH) in Escherichia coli. Isocitrate and calcium participate in the binding of the enzyme, with the calcium competitively preventing magnesium and resulting in the total prevention of turnover. The process of turnover necessitates the embedding of the isocitrate molecule in the active-site aperture by the nicotinamide ring. Isocitrate binding is followed by NADP+, revealing an organized binding in a series mechanism.

The molecular recognition mechanisms undergone by isocitrate dehydrogenase (IDH) and and maltose binding protein with their substrates were discussed. The results of computational simulations and actual analysis of enzymatic or protein-protein interactions showed that both substrate and substrate analogs can dock to the active site of IDH. In addition, during the chemotatic response to maltose, the MBP binds to the extracellular domain of the maltose receptor. The results demonstrate how computational simulations can be combined to structural and energy data to provide information neccessary for the prediction of complex binding events.

A study was conducted on the transmembrane signaling of the Escherichia coli aspartate receptor. The first membrane region's (TTM1) cytosolic extension was structurally characterized along with its conformational changes. Results showed that TM1's cytosolic extension is helical by nature and that the helix is more solvent-exposed at the membrane's cytosilic side. Results also showed that aspartate binding increases the rate of cross-linking.

The roles of cAMPP and calcium in the habituation response of PC12 cells were investigated. The results showed that cAMP could increase norepinephrine secretion in response to repetitive stimuli such as depolarization and acetylcholine. However, the pathway by which cAMP increases norepinephrine secretion is distinct from that controlling internal calcium levels, which are reflective of the habituation response. A model for the calcium-dependent modulation of norepinephrine secretion and cAMP metabolism isproposed.

Neuronally differentiated PC12 cells were repeatedly stimulated with either acetylcholine (ACh) or ATP todetermine if these cells would exhibit characteristics of long-term or short-term memory after habituation. The results showed that norepinephrine release declined exponentially in response to repeated stimuli of either ACh orATP. In addition, after habituation recovery, the response to a second series of stimuli declined more rapidly. These results demonstrate that habituation ofPC12 cells could result in responses characteristic of short-term and long-termmemory.

The NtrC protein is the response regulator in the signal transduction pathway activated during nitrogen starvation in Escherichia coli and Salmonella typhimurium. Its transactivating activity requires a transient phosphorylated state. The site of phosphorylation of the S. typhimurium NtrC protein was determined. The results showed that this site is Asp-54, within the N-terminal protein-phosphatase domain. Phosphorylation of this site results in the allosteric enhancement of transactivation by the C-terminal region of NtrC.

The binding sites of a maltose-binding protein (MBP) can be anticipated by a 'binary docking' method involving two MBP octapeptide sequences with mutations that prevent maltose chemotaxis. The peptides in the docked solution permit the construction of an MBP-receptor complex. The dependability of this technique gives it great promise in foreseeing protein-protein and peptide-protein interactions, a necessary step in drug design.

A study was carried out on a series of mutants of chymotrypsin inhibitor 2 (Cl2) to determine their relative importance for inhibition of the serine protease subtilisin BPN', and for resistance of the inhibitor to proteolysis. The above-mentioned properties were investigated in tandem with the crystal structure of the mutant inhibitor-enzyme complexes

A series of mutants of chymotrypsin inhibitor 2 (C12), at residues that interact with the inhibited enzyme subtilisin BPN' were studied to determine the relative importance of intermolecular contacts on either side of the scissile bond. The crystal structures along with biochemical data support a transition-state stabilization role for the P(sub 1) residue in subtilisin catalysis.

Research has been conducted on synthetic cyclic peptides which mimic the binding loop of chymotrypsin inhibitor 2. Results demonstrate that the peptides have been cleaved by subtilisin, and that in the absence of the inhibitor's structural context the sequence provides specificity for the the bond hydrolysis.

Poly-[Alpha]2,8-sialic acid (PSA) has been implicated in numerous normal and pathological processes, including development, neuronal plasticity, and tumor metastasis. We report that cell surface PSA expression can be reversibly inhibited by a small molecule, N-butanoylmannosamine (ManBut). Inhibition occurs through a metabolic mechanism in which ManBut is converted to unnatural sialic acid derivatives that effectively act as chain terminators during cellular PSA biosynthesis. N-Propanoylmannosamine (HanProp), which differs from ManBut by a single methylene group, did not inhibit PSA biosynthesis. Modulation of PSA expression by chemical means has a role complementary to genetic and biochemical approaches in the study of complex PSA-mediated events., The biological functions of cell surface oligosaccharides have been difficult to elucidate owing to the complexity of achieving genetic control over a molecule that is the product of multiple enzymes [...]

The problem of the propagation of conformational changes over long distances or through a closely packed protein is shown to fit a model of a ligand-induced conformational change between two protein states selected by evolution. Moreover, the kinetics of the pathway between these states is also selected so that the energy of ligand binding and the speed of the transition between conformational states are physiologically appropriate. The crystallographic data of a wild-type aspartate receptor that has negative cooperativity and a mutant that has no cooperativity but has native transmembrane signaling are shown to support this model.

Science. Dr. Noitall, you are the world authority on elections, the man who invented the electoral college, the man who showed how tombstones could vote, the man who created the [...]

To characterize the mechanism by which receptors propagate conformational changes across membranes, nitroxide spin labels were attached at strategic positions in the bacterial aspartate receptor. By collecting the electron paramagnetic resonance spectra of these labeled receptors in the presence and absence of the ligand aspartate, ligand binding was shown to generate an ~1 angstrom intrasubunit piston-type movement of one transrnembrane helix downward relative to the other transmembrane helix. The receptor-associated phosphorylation cascade proteins CheA and Chew did not alter the ligand-induced movement. Because the piston movement is very small, the ability of receptors to produce large outcomes in response to stimuli is caused by the ability of the receptor-coupled enzymes to detect small changes in the conformation of the receptor., Cells receive signals from the outside world by way of receptors that span the membrane. Although some receptors transmit information across the membrane by means of an ion channel that [...]

On page 895 of this issue, Ferrell and Machleder[1] highlight a new era in our understanding of cellular metabolism. Knowledge of metabolic processes in cells can be roughly divided into […]

Author(s): Daniel E. Koshland, Jr [1] Conventional wisdom suggests that it may take as long as 30 years from the 'eureka' of a basic medical research discovery to the availability [...]

Glenn Seaborg had a career that few will emulate in any millennium. He discovered an element that ended a war and prevented more wars among the major powers for 50 [...]