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The crystal structure of a complex between a phosphonate transition-state analogue and the phospholipase A2 (PLA2) from Naja naja atra venom has been solved and refined to a resolution of 2.0 angstroms. The identical stereochemistry of the two complexes that comprise the crystal's asymmetric unit indicates both the manner in which the transition state is stabilized and how the hydrophobic fatty acyl chains of the substrate are accommodated by the enzyme during interfacial catalysis. The critical features that suggest the chemistry of binding and catalysis are the same as those seen in the crystal structure of a similar complex formed with the evolutionarily distant bee-venom PLA2.

Ras proteins are post-translationally modified by farnesylation. In the present investigation, we identified an activity in crude soluble extracts of yeast cells that catalyzes the transfer of a farnesyl moiety from farnesyl pyrophosphate to yeast RAS2 protein. RAS2 proteins having a C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid and Xaa is the unspecified C-terminal amino acid) served as substrates for this reaction, whereas RAS2 proteins with an altered or deleted Cys-Ali-Ali-Xaa sequence did not. A yeast mutant, dpr1/ram1, originally isolated as a Ras-processing mutant was shown to be defective in farnesyltransferase activity. In addition, another mutant, ram2, also was defective in the transferase activity. These results demonstrate that at least two genes, DPR1/RAM1 and RAM2, are required for the farnesyltransferase activity in yeast.

We have shown previously that guanine nucleotide-binding protein (G protein) beta gamma complexes purified from bovine brain membranes are methyl esterified on a C-terminal cysteine residue of the gamma polypeptide. In the present study, 3H-methylated G beta gamma complexes cleaved to their constituent amino acids by exhaustive proteolysis were shown to contain radiolabeled material that coeluted with geranylgeranylcysteine methyl ester on reversed-phase HPLC and two TLC systems. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the prenyl modification occurs on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G beta gamma by treatment with Raney nickel positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. To delineate the distribution of this modification among gamma subunits, purified G beta gamma complexes were separated into 5-kDa (gamma 5) and 6-kDa (gamma 6) forms of the gamma polypeptide by reversed-phase HPLC. Gas chromatography-coupled mass spectrometry analyses of Raney nickel-treated purified gamma 5 and gamma 6 subunits showed that both polypeptides were modified by geranylgeranylation. These results demonstrate that at least two forms of brain gamma subunit are posttranslationally modified by geranylgeranylation and carboxyl methylation. These modifications may be important for targeting G beta gamma complexes to membranes.

The action of the phospholipases A2 (PLA2s) from Naja naja naja, Naja naja atra, and Crotalus atrox venoms as well as the enzyme from porcine pancreas on a number of short-chain, water-soluble substrates was studied. The inhibition of these enzymes by short-chain phosphonate- and thiophosphonate-containing phospholipid analogues was also examined. The kinetic patterns observed for the action of the venom PLA2s on substrates containing phosphocholine head groups all deviated from a classical Michaelis-Menten-type behavior. With a substrate containing an anionic head group, the kinetic pattern observed was more normal. In contrast, Michaelis-Menten-type behavior was observed for the action of the porcine pancreatic PLA2 acting on all of the substrates studied. A short-chain phospholipid analogue in which the enzyme-susceptible ester was replaced with a phosphonate group was found to be a tight-binding inhibitor of the venom PLA2s with IC50 values that were some 10(4)-10(5)-fold lower than the concentration of substrate used in the assay. The degree of inhibition was found to depend dramatically on the stereochemical arrangement of substituents in the inhibitor which strongly suggests that the inhibitors are binding directly to the active site of the PLA2s. By comparison, the phosphonate analogue functioned as a poor inhibitor of the porcine pancreatic PLA2. Direct inhibitor binding studies indicated that the short-chain phosphonate inhibitor bound weakly to the venom enzymes in the absence of the short-chain substrates. Several other unusual features of the inhibition were also observed. The data are interpreted in terms of a model in which the enzyme and substrate form a lipid-protein aggregate at substrate concentrations below the critical micelle concentration (cmc). Possible reasons for the selective binding of the inhibitor to the enzyme-substrate microaggregate are discussed.

Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.

Chagas disease results from infection with the protozoan pathogen Trypanosoma cruzi ( T. cruzi ). The disease is endemic in South and Central America with > 16 million people chronically infected and ≈14,000 deaths per year. Current treatment options are inadequate to cure this infection; thus, research is needed to discover better chemotherapeutics. We have an ongoing project making inhibitors to a key enzyme involved in sterol biosynthesis of T. cruzi , sterol 14-demethylase (Tc14DM). This enzyme, the target of azole antifungal drugs, is a well established antimicrobial target. We hypothesize that new compounds can be synthesized with optimal anti- T. cruzi activity, and these will be sufficiently active to cure animals (and humans) with chronic T. cruzi infection. We have discovered two new chemical classes that bind the Tc14DM and that have potent activity against T. cruzi cultures with ED 50 values in the 1-10 nM range. The active compounds are a series of disubstituted imidazoles and another series derived from the cancer drug tipifarnib. We have shown the disubstituted imidazoles dramatically suppress T. cruzi parasitemia in the mouse model of Chagas disease. Working in collaboration with chemists at Yale University and University of Washington, we are working to optimize these lead compounds for pharmacological properties and efficacy against T. cruzi .

We have previously identified proteolytic activity in rat liver microsomes that cleaves an intact tripeptide, VIS, from S-farnesylated-CVIS tetrapeptide. This enzymatic activity, termed prenyl protein-specific endoprotease (PPEP) activity, has been solubilized in CHAPS and purified 5-fold. To probe the peptide recognition features of PPEP activity, 64 tripeptides [N-acetyl-C(S-farnesyl)a1a2] were prepared and tested as competitive inhibitors of PPEP activity-catalyzed hydrolysis of N-acetyl-C(S-farnesyl)VI[3H]S. It was found that PPEP activity prefers large hydrophobic residues in the a1 and a2 positions. A subset of N-acetyl-C(S-farnesyl)a1a2 peptides were prepared in radiolabeled form, and it was found that PPEP activity preferences for these substrates correlated well in most cases with the inhibition data. The exception is that R in the a1 position does not prevent binding of peptide to PPEP activity, but such peptides are poor substrates. The anionic residue D in the a2 position is not tolerated by PPEP activity. Five farnesylated radiolabeled tetrapeptides, Ac-C(F)FM[3H]L, Ac-C(F)LI[3H]L, Ac-C(F)LL[3H]L, Ac-C(F)LM[3H]L, and Ac-C(F)VI[3H]L were prepared, and PPEP activity kinetic studies revealed that they are good substrates and show comparable KM values (2.2-13.5 microM). Ac-C(F)RL[3H]S is a poor substrate. The reported peptide binding preferences of PPEP activity should be useful in designing compounds that block the C-terminal proteolysis of prenylated proteins. Nonprenylated peptides do not bind to PPEP activity, and replacement of the farnesyl group with ann-pentadecyl group modestly reduces binding. Peptide-membrane partitioning studies were used together with theoretical arguments to fully understand the substrate specificity of PPEP activity toward these compounds.

Many signaling proteins, including the proto-oncogene Ras, require the covalent addition of a lipid chain for proper function. This prenylated protein is then earmarked for further proteolysis and methylation. In his Perspective, Gelb discusses two reports in this issue that illuminate how the lipid is initially attached to the protein [Park et al . ( p. 1800 )] and how the later proteolytic modifications are accomplished [Boyartchuk et al . ( p. 1796 )]

Bacterial cytochrome P450cam catalyzes the hydroxylation of camphor to yield 5-exo-hydroxycamphor in vivo and in a reconstituted system with oxygen, pyridine nucleotide, flavoprotein dehydrogenase, and putidaredoxin. Product is also formed when the ferric form of the hemoprotein is mixed with the exogenous oxidants iodosobenzene, m-chloroperbenzoic acid, and hydrogen peroxide. In this paper we show that when the P450cam-dependent hydroxylation reactions are studied with camphor analogues containing deuterium at either the 5-exo or 5-endo position, a very small intermolecular isotope on the overall reaction velocity is observed and a significant intramolecular isotope effect is documented. We suggest the existence of an intermediate substrate-carbon radical and demonstrate that abstraction can occur from either the exo or endo position at carbon 5 on the camphor skeleton, with the oxygen stereospecifically added to only the Re face to give 5-exo-hydroxycamphor as the unique product. Using these substrates, we observed nearly identical hydrogen/deuterium isotope ratios in the product alcohol for the pyridine nucleotide/atmospheric dioxygen as well as exogenous oxidant supported hydroxylations, suggesting that these reactions share a common hydrogen-abstracting species. The relatively small magnitude of the measured intramolecular isotope effect can be rationalized with a model involving a reversible hydrogen-abstraction step and/or the involvement of heavy-atom motion in the reaction coordinate.

The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.

We recently showed that HeLa cell lamin B is modified by a mevalonic acid derivative. Here we identified the modified amino acid, determined its mode of linkage to the mevalonic acid derivative, and established the derivative′s structure. A cysteine residue is modified because experiments with lamin B that had been biosynthetically labeled with [3H]mevalonic acid or [35S]cysteine and then extensively digested with proteases yielded 3H- or 35S-labeled products that co-chromatographed in five successive systems. A thioether linkage rather than a thioester linkage is involved because the mevalonic acid derivative could be released from the 3H-labeled products in a pentane-extractable form by treatment with Raney nickel but not with methanolic KOH. The derivative is a farnesyl moiety because the Raney nickel-released material was identified as 2,6,10-trimethyl-2,6,10-dodecatriene by a combination of gas chromatography and mass spectrometry. The thioether-modified cysteine residue appears to be located near the carboxyl end of lamin B because treatment of 3H-labeled lamin B with cyanogen bromide yielded a single labeled polypeptide that mapped toward this end of the cDNA-inferred sequence of human lamin B.

Derivatives of isatoic anhydride were prepared and tested as inhibitors of serine proteases. A number of isatoic anhydrides with positively charged substituents irreversibly inactivated several trypsin-like enzymes and preferentially inactivated trypsin over chymotrypsin. Further selectivity was obtained by introduction of an aromatic group on the N-1 position of isatoic anhydride. 7-(Aminomethyl)-1-benzylisatoic anhydride was prepared and was a selective inactivator of thrombin; thus it is possible to prepare derivatives of isatoic anhydride that are highly enzyme selective without attaching peptide recognition structures.

Synthese d'analogues de phosphatidylcholine ou le groupe situe en 2 est remplace par des oxo-2 alkyle ou des hydroxy-2 alkyle

The mechanism of inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone has been studied. Chloride analysis of the inactivated enzyme suggests that the complex does not contain intact chloropyrone or an acid chloride. 13C NMR studies of the enzyme inactivated with 13C-enriched chloropyrones show that (1) the pyrone ring is no longer intact, (2) C-6 becomes a carboxylate group and C-2 becomes esterified to the enzyme, probably to serine-195, and (3) a double bond is present adjacent to the serine ester. The inactivated enzyme slowly regains catalytic activity with the concomitant release of (E)-4-benzyl-2-pentenedioic acid. It is concluded that double bond migration occurs during reactivation since the position of the double bond in the released diacid product is different than in the inactivator-enzyme complex. When the reactivation is carried out in [18O]H2O-enriched water, a single oxygen-18 is incorporated into the released product and is further evidence that the inactivator is bound to the enzyme only through a single ester linkage. A deuterium isotope effect on reactivation is observed when a chloropyrone deuterated at C-5 is used. This result demonstrates that removal of a proton from C-5 is required for reactivation and that isomerization of the double bond and not hydrolysis of the acyl-enzyme is rate determining. A variety of amines accelerate the rate of reactivation by functioning as general bases and not as nucleophiles. A reaction scheme is presented that accounts for the formation of the stable inactivator-enzyme complex as well as the production of two products derived from enzymatic hydrolysis of the chloropyrone.(ABSTRACT TRUNCATED AT 250 WORDS)

The enzyme N-succinyl-L-diaminopimelic acid desuccinylase from Escherichia coli has been purified 7,100-fold to apparent homogeneity. The enzyme is part of the diaminopimelic acid-lysine pathway in bacteria and catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to produce L-diaminopimelic acid and succinate. The enzyme exists as a mixture of dimeric and tetrameric species of identical subunits of molecular weight approximately 40,000. Activity was completely abolished following dialysis of the enzyme against metal chelators. Cobalt(II) and zinc were effective in restoring the activity. The apparent affinities of the apoenzyme for cobalt and zinc were similar (Kd values near 1 microM) and the cobalt enzyme was 2.2-fold more active than the zinc enzyme. The Km and turnover number for the hydrolysis of the natural substrate, N-succinyl-L-diaminopimelic acid, were 0.4 mM and 16,000 min-1, respectively. The substrate specificity of the enzyme was defined by preparing a number of substrate analogues that systematically lack the various functional groups present in the molecule. These studies show that the enzyme is highly specific for the natural substrate. These properties of N-succinyl-L-diaminopimelic acid desuccinylase and the fact that the enzyme is essential for bacterial growth make it an ideal target for the development of inhibitors with potential antibacterial activity.

The inactivation of chymotrypsin by 5-benzyl-6-chloro-2-pyrone has been studied. Chloride analysis of the inactivated enzyme suggests that chlorine is no longer present in the complex. 13C NMR spectroscopy of chymotrypsin inactivated with 5-benzyl-6-chloro-2-pyrone-2,6-13 C2 shows the presence of two new resonances from the protein-bound inactivator. The chemical shift values of these resonances are consistent with an intact pyrone ring on the enzyme as well as the replacement of the C-6 chlorine by a different heteroatom. X-ray diffraction analysis at 1.5-A resolution of the inactivator-enzyme complex demonstrates that the gamma-oxygen of the active site serine residue (serine 195) is covalently attached to C-6 of the inactivator and that the pyrone ring is intact. The 5-benzyl group of the inactivator is bound to the enzyme in the hydrophobic specificity pocket. The conformational changes that occur in the protein as a result of complexation with the inactivator are discussed.

In this study the membrane orientation of a tryptophan-flanked model peptide, WALP23, was determined by using peptides that were labeled at different positions along the sequence with the environmentally sensitive fluorescent label BADAN. The fluorescence properties, reflecting the local polarity, were used to determine the tilt and rotation angles of the peptide based on an ideal alpha-helix model. For WALP23 inserted in dioleoylphosphatidylcholine (DOPC), an estimated tilt angle of the helix with respect to the bilayer normal of 24 degrees +/- 5 degrees was obtained. When the peptides were inserted into bilayers with different acyl chain lengths or containing different concentrations of cholesterol, small changes in tilt angle were observed as response to hydrophobic mismatch, whereas the rotation angle appeared to be independent of lipid composition. In all cases, the tilt angles were significantly larger than those previously determined from (2)H NMR experiments, supporting recent suggestions that the relatively long timescale of (2)H NMR measurements may result in an underestimation of tilt angles due to partial motional averaging. It is concluded that although the fluorescence technique has a rather low resolution and limited accuracy, it can be used to resolve the discrepancies observed between previous (2)H NMR experiments and molecular-dynamics simulations.

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.

In this data article we provide a detailed standard operating procedure for performing a tandem mass spectrometry, multiplex assay of 6 lysosomal enzymes for newborn screening of the lysosomal storage diseases Mucopolysaccharidosis-I, Pompe, Fabry, Niemann-Pick-A/B, Gaucher, and Krabbe, (Elliott, et al., 2016) [1]. We also provide the mass spectrometry peak areas for the product and internal standard ions typically observed with a dried blood spot punch from a random newborn, and we provide the daily variation of the daily mean activities for all 6 enzymes.

Derivatives of isatoic anhydride were prepared and tested as inhibitors of serine proteases. A number of isatoic anhydrides with positively charged substituents irreversibly inactivated several trypsin-like enzymes and preferentially inactivated trypsin over chymotrypsin. Further selectivity was obtained by introduction of an aromatic group on the N-1 position of isatoic anhydride. 7-(Aminomethyl)-1-benzylisatoic anhydride was prepared and was a selective inactivator of thrombin; thus it is possible to prepare derivatives of isatoic anhydride that are highly enzyme selective without attaching peptide recognition structures.

The iron transport protein, transferrin, binds two metal ions and, concomitantly, two carboxylate anions. The metal ion indicators, xylenol orange and semi-xylenol orange are carboxylate anions which exhibit a characteristic visible spectrum when attached to a metal. We prepared the ternary complexes VO2+-transferrin-xylenol orange and VO2+-transferrin-semi-xylenol orange. The EPR spectra show that the vanadyl ion is attached to the protein and the visible spectra show that the xylenol orange or semi-xylenol orange is attached to the metal. The implication is that in other metal-transferrin-anion complexes, the anion is directly attached to the metal.

Kinetic studies with phospholipase A2 are complicated by the fact that binding of the enzyme to the interface precedes catalytic turnover. This difficulty can be overcome by monitoring interfacial catalysis in the scooting mode where the enzyme does not leave the interface. The kinetics of inhibition by transition-state analogues shows that specific competitive inhibition is the result of competition between inhibitor and substrate for the binding to the active site of the enzyme in the interface. Several lipophilic compounds, including alkanols, substituted butyrophenones, aristolochic acid, and mepacrine apparently reduce the rate of lipolysis by promoting the desorption of phospholipase A2 from the interface.

In addition to the normal 5- exo -hydroxylation of camphor, bacterial cytochrome P450 is shown to carry out the facile epoxidation of dehydrocamphor to give exo -5,6-epoxycamphor. A detailed kinetic study of the reaction demonstrates that epoxidation and hydroxylation reactions occur with nearly identical rates both in the reconstituted system containing flavoprotein dehydrogenase, iron-sulfur protein, and NADH as well as in the single turnover reaction beginning with ferrous, oxygenated cytochrome P450. Dehydrocamphor is not a suicide substrate for the enzyme since competent enzyme remains after several thousand reaction cycles per P450 molecule.

A variety of metal ions can bind to the iron-transport protein, transferrin, at two specific sites. For each metal ion, a carboxylate anion is concomitantly bound. Six metal ions which were examined fall into two classes based on proton release and ultraviolet spectral changes which accompany binding to the protein. Class II ions, which include Cu2+ and Zn2+, release approximately 2 H+/metal bond. Class III ions, which include Fe3+, Ga3+, Al3+, and VO2+, release approximately 3 H+/metal bound. The increase in absorbance near 242 nm, characteristic of tyrosine ionization, has the ratio 0.55–0.75 for class II:class III ions. Both Fe3+ and Cu2+ form metal-transferrin-oxalate complexes in the presence of excess C2O42−. Fe3+ releases close to 3 H+/metal whether forming oxalate or bicarbonate complexes with transferrin. Binding of Cu2+ to transferrin releases 2 H+/metal in the presence of C2O2−4 or HCO3−. Since equal numbers of H+/metal are released for both anions, it is likely that the bicarbonate ion does not lose its proton, and remains as HCO3− in transferrin. These results are interpreted in terms of possible combinations of ligands at the metal binding sites.

The pentapeptide BOC-Phe-Phe-difluorostatone-Leu-Phe-NH2 has been prepared and found to be a potent inhibitor of human renin. This compound contains a difluoromethylene ketone group that exists predominantly in the hydrated form in water. The difluorostatone-containing peptide is 7-fold and 22-fold more potent than the analogous statine- and statone-containing peptides, respectively. Structure/activity analysis of the most potent inhibitor was carried out by replacing some of the peptide bonds with trans-alkenes. In all cases, a dramatic loss in binding to renin was observed. A number of statine-containing inhibitors of renin have been reported and this work suggests that the replacement of statine with difluorostatone will yield more potent compounds.

Bacterial cytochrome P-450 induced by camphor (P-450cam) is reconstituted with manganese-protoporphyrin IX, yielding an enzyme that displays unique spectral properties relative to previously characterized manganese-porphyrin systems. The nitric oxide complex of the manganese(II)-protein shows a hyper-metalloporphyrin spectrum suggestive of thiolate ligation to the porphyrin-bound manganese ion. In the presence of iodosobenzene as a source of active oxygen, manganese-substituted cytochrome P-450cam serves as a catalyst for the epoxidation of an enzyme-bound olefin substrate. This reactivity proceeds through a spectrally detectable intermediate that resembles the manganese(V)-oxo complexes that have been well documented with model systems employing artificial manganese-metalloporphyrins in organic solution. Interestingly, manganese-substituted cytochrome-P-450cam shows no hydroxylation activity either in the reconstituted camphor hydroxylase system with pyridine nucleotide or in the presence of iodosobenzene and the Mn(III) form of the protein.