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M H Gelb

Publications and source records attributed to M H Gelb.

At least 109 records · Page 6Linked to original sources
PubMed1995

Interfacial enzymology of glycerolipid hydrolases: lessons from secreted phospholipases A2.

Interfacial enzymes operate at an organized interface such as lipid aggregates in contact with the aqueous phase. The enzyme phospholipase A2 is a well studied interfacial enzyme, and a discussion of its behavior at interfaces is the topic of this review. Knowledge gained from studies of phospholipases A2 can be applied toward the quantitative analysis of other interfacial enzymes. The kinetic analysis of these enzymes is greatly simplified if one establishes certain experimental conditions that limit the exchange of enzyme and substrate between different substrate aggregates. With such constraints, the kinetics can be analyzed in terms of classical Michaelis-Menten theory adopted for the action of enzymes at interfaces. It is also possible to describe other enzyme properties such as inhibition and substrate preferences in a meaningful way using formalism that is well known in solution-phase enzymology.

Animals↗
PubMed1994

Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A.

Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini. They differ from other geranylgeranylated small GTPases in several important respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs: -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. We addressed this question by direct structural analysis of in vitro prenylated proteins. We incubated recombinant Rab geranylgeranyltransferase, Rab escort protein, and [1-3H]geranylgeranyl pyrophosphate with recombinant wild-type Rab1A (-XXCC), Rab3A (-XCXC), or Rab5A (-CCXX) and treated each labeled protein with trypsin. We then analyzed the resulting peptides by HPLC and electrospray mass spectrometry and found that for each protein both C-terminal adjacent cysteines were geranylgeranylated. These results indicate that Rab geranylgeranyltransferase/Rab escort protein catalyzes the geranylgeranylation of both cysteines in Rab proteins with three distinct C-terminal motifs and suggest that other Rab proteins with these motifs may be similarly modified.

Alkyl and Aryl Transferases↗
PubMed1994

Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombin-stimulated human platelets.

An analogue of arachidonic acid in which the COOH group is replaced by a trifluoromethyl ketone group (COCF3) has recently been shown to be a tight binding inhibitor of the 85-kDa cytosolic phospholipase A2 that is found in platelets and other cells (Street, I. P., Lin, H.-K., Laliberté, F., Ghomashchi, F. G., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940). This trifluoromethyl ketone inhibits most of the arachidonate release from the phospholipid pool in thrombin-stimulated human platelets at concentrations of 0-40 microM with 4 x 10(8) platelets/ml. A structure-function analysis of related compounds reveals a good correlation between the inhibition of the purified phospholipase A2 and the blockage of arachidonate release in platelets. A number of recently described potent inhibitors of the 14-kDa phospholipase A2 that is secreted from activated platelets have no effect on the level of free arachidonate production. Furthermore, the addition of a large amount of recombinant 14-kDa phospholipase A2 to platelets does not produce free arachidonate, and it does not alter the amount of arachidonate released following platelet activation with thrombin. These studies provide strong pharmacological evidence for the role of the cytosolic phospholipase A2 in producing most, if not all, of the liberated arachidonate in thrombin-stimulated human platelets, and they show that tight binding membrane-residing inhibitors of the cytosolic phospholipase A2 can block the eicosanoid cascade in living cells.

Arachidonic Acids↗
PubMed1994

Antigenic surface of the bee venom allergen phospholipase A2. Structural functional analysis of human IgG4 antibodies reveals potential role in protection.

In the human immune response to the main bee venom allergen phospholipase A2 (PLA2), Abs of the IgG4 isotype are associated with protection. The antigenic sites of PLA2 that are recognized by two mAb of this isotype have been analyzed at the molecular level. The interaction of PLA2 with these two Abs has previously been shown to depend on the conformation of the Ag and to be sensitive to lysine modifications. Therefore, genetically engineered Ag displaying single point mutations of lysine residues were generated. Nine out of twelve surface-exposed lysine residues were substituted by glutamate or glutamine, and the effect of individual mutations on mAb binding was analyzed in the context of the folded Ag. Substitution of lysine at position 25 with either glutamate or glutamine completely abrogates binding to both mAb. All other mutants do not show a difference in binding when compared with wild-type Ag. Probing of sera from bee keepers with lysine 25 mutants reveals that a significant proportion of serum Abs of the IgG4 isotype display specificity for this epitope, suggesting that this represents a major B cell-antigenic site in hyperimmune individuals. We further found that both mAbs inhibit the catalytic activity of PLA2, a property that may account for the protective role of IgG4 Abs in hyperimmune individuals.

Allergens↗
PubMed1994

Characterization of interfacial catalysis by Aeromonas hydrophila lipase/acyltransferase in the highly processive scooting mode.

A glycerophospholipid:cholesterol acyltransferase (GCAT) that also has lipase activity is secreted by the bacterium Aeromonas hydrophila. Hydrolysis of the sn-2-ester bond of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) vesicles by this enzyme is shown to occur in a highly processive scooting mode in which the enzyme, substrate, and the products of hydrolysis remain bound to the vesicle interface. This conclusion is based on the following observations. (a) When there is an excess of vesicles over enzyme, the hydrolysis of the sn-2-acyl group ceases after only a fraction of the total available substrate is hydrolyzed. Addition of more enzyme, but not of more substrate, leads to a new round of hydrolysis. (b) The extent of hydrolysis of vesicles per enzyme increases with the size of the vesicles, and it corresponds to the total hydrolysis of the outer monolayer of one vesicle by one enzyme. (c) The enzyme bound to vesicles composed of reaction products or of the non-hydrolyzable phospholipid 1,2-ditetradecyl-sn-glycero-3-phosphomethanol (DTPM) is not able to undergo intervesicle exchange. Instead, intervesicle transfer of the substrate or the bound enzyme due to vesicle fusion promotes hydrolysis of all of the vesicles present in the reaction mixture. (d) Addition of DTPM vesicles to a reaction mixture containing DMPM substrate vesicles and the enzyme has no noticeable effect on the course of hydrolysis. Substrate specificity studies in the scooting mode on DMPM vesicles reveal that GCAT displays essentially no selectivity in the hydrolysis of phospholipids with different polar head groups. Treatment of GCAT with trypsin, which removes a small peptide, results in an enzyme that displays comparable catalytic activity but increased affinity for the interface. Alkyltrifluoromethyl ketones are shown to be tight-binding competitive inhibitors of GCAT. The scooting mode analysis, which has previously been shown to provide a simplified approach for analyzing the steady-state kinetics of interfacial catalysis by secreted phospholipase A2, is also useful for analyzing the interfacial kinetic behavior of lipases.

Acyltransferases↗
PubMed1994

Inhibition of phospholipase A2.

Phospholipases A2 are involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Inhibitors of these enzymes are proving useful in determining the biological roles of phospholipases A2 in complex cellular processes and may also have therapeutic potential. Inhibition of these lipolytic enzymes is more difficult to characterize as the enzymatic reaction occurs at a lipid/water interface. This review focuses on the description of a number of classes of rationally designed phospholipase A2 inhibitors. The development of a theoretical framework for the proper analysis of inhibitors is presented. Structural studies of phospholipase A2-inhibitor complexes suggest how the lipolysis reaction is catalyzed. Finally, some recent results on the use of phospholipase A2 inhibitors in living cells and tissues are revealed.

Animals↗
PubMed1994

Lysine residues in bee venom phospholipase A2 are important for binding to human monoclonal or polyclonal antibodies of the IgG4 isotope.

Discontinuous antigenic sites on bee venom phospholipase A2 (PLA) have been mapped using human monoclonal antibodies or human polyclonal serum antibodies (hpAbs) of the IgG4 isotype from beekeepers or of the IgE isotype from individuals allergic to PLA. Lysine residues of PLA have been specifically modified by acetylation or acylation by treatment with citraconic anhydride of their epsilon-amino groups to analyze their role in antigen-antibody binding. After the modifications, the binding of PLA to the human monoclonal antibodies is lost, whereas the binding to IgG4 hpAbs is significantly decreased. In contrast, the effect on the binding of PLA to IgE hpAbs appears to be more heterogeneous. The data indicate the importance of lysine residues as being part of B cell epitopes in PLA-specific antibodies of the IgG4 isotype, but less so for those of the IgE isotype.

Acetylation↗
PubMed1994

Interleukin 1 alpha causes rapid activation of cytosolic phospholipase A2 by phosphorylation in rat mesangial cells.

We have shown previously that interleukin 1 (IL-1) stimulates eicosanoid production in glomerular mesangial cells (MC) by de novo synthesis of a 14-kD, group II phospholipase A2 (PLA2). IL-1-stimulated prostaglandin E2 synthesis precedes expression of this enzyme, suggesting that another PLA2 isoform must be more rapidly activated. In the presence but not absence of calcium inophore, [3H]arachidonate release is increased significantly as early as 5 min after addition of IL-1, and IL-1 concurrently stimulates a Ca(2+)-dependent phospholipase activity, which was characterized as the cytosolic form of PLA2 (cPLA2). IL-1 does not alter either cPLA2 mRNA expression or mass in serum-stimulated MC, suggesting that cPLA2 activity is increased by a posttranslational modification. IL-1 treatment for 30 min doubles 32P incorporation into immunoprecipitable cPLA2 protein, concordant with the increase in enzyme activity. Immunoblot analysis of extracts derived from IL-1-treated (30 min) cells demonstrates a decreased mobility of cPLA2, and treatment of MC lysates with acid phosphatase significantly reduces cytokine-activated cPLA2 activity, further indicating that IL-1 stimulates phosphorylation of the enzyme. IL-1 treatment (24 h) of serum-deprived MC doubled cPLA2 mRNA, protein, and activity. In summary, IL-1 increases cPLA2 activity in a biphasic, time-dependent manner both by posttranslational modification and de novo synthesis. We consider cPLA2 activation a key step in IL-1-stimulated synthesis of pro-inflammatory, lipid mediators, and an integral event in the phenotypic responses induced in target cells by this cytokine.

Animals↗
PubMed1993

A prenylated protein-specific endoprotease in rat liver microsomes that produces a carboxyl-terminal tripeptide.

The maturation of proteins that contain the C-terminal sequence Cys-Ali-Ali-Xaa (where Ali is usually an aliphatic amino acid and Xaa is a number of different amino acids) involves the attachment of a farnesyl or geranylgeranyl group to the cysteine residue, proteolytic removal of the C-terminal three amino acids, and methylation of the prenylated cysteine alpha-carboxyl group. Two prenylated and radiolabeled peptides were prepared in order to detect the proteolysis step(s) in a cell-free system and to determine the reaction products. These peptides are ECB-NPFRQRRFFC(S-geranylgeranyl)AI[3H]L and ECB-C(S-farnesyl)VI[3H]S (ECB is an extended-chain biotin group) which are patterned after the C-termini of geranylgeranylated and farnesylated G protein gamma-subunits. Incubation of these peptides with rat liver microsomes, but not cytosol, results in the production of radiolabeled dipeptides (I[3H]L and I[3H]S) and tripeptides (AI[3H]L and VI[3H]S) as the major products and smaller amounts of amino acids ([3H]L and [3H]S). A multitude of independent experiments shows that the dipeptides are produced from the tripeptides by secondary proteolysis. Although a portion of the [3H]S produced comes directly from ECB-C(S-farnesyl)VI[3H]S, the KM of > 30 microM for this reaction is significantly higher than the KM of 1.1 microM for the production of VI[3H]S from the farnesylated peptide. This suggests that the carboxypeptidase is not part of the pathway for the maturation of prenylated proteins. Nonprenylated peptides at concentrations of 10-100-fold higher than those of the prenylated substrates did not reduce the amount of tripeptide produced.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2.

A trifluoromethyl ketone analogue of arachidonic acid in which the COOH group is replaced with COCF3 (AACOCF3) was prepared and found to be a tight- and slow-binding inhibitor of the 85-kDa cytosolic human phospholipase A2 (cPLA2). Enzyme inhibition was observed when AACOCF3 was tested in assays using either phospholipid vesicles or phospholipid/Triton X-100 mixed micelles. The fact that the inhibition developed over several minutes in both assays establishes that AACOCF3 inhibits by direct binding to the enzyme rather than by decreasing the fraction of enzyme bound to the substrate interface. From the measured values of the inhibitor association and dissociation rate constants, an upper limit of the equilibrium dissociation constant for the Ca(2+).AACOCF3.PLA2 complex of 5 x 10(-5) mole fraction was obtained. Thus, detectable inhibition of cPLA2 by AACOCF3 occurs when this compound is present in the assay at a level of one inhibitor per several thousand substrates. Arachidonic acid analogues in which the COOH group is replaced by COCH3, CH(OH)CF3, CHO, or CONH2 did not detectably inhibit the cPLA2. The arachidonyl ketones AACOCF2CF3 and AACOCF2Cl were found by 19F NMR to be less hydrated than AACOCF3 in phospholipid/Triton X-100 mixed micelles, and compared to AACOCF3 these compounds are also weaker inhibitors of cPLA2. In keeping with the fact that cPLA2 displays substrate specificity for arachidonyl-containing phospholipids, the arachidic acid analogue C19H39COCF3 is a considerably less potent inhibitor compared to AACOCF3.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic Acids↗
PubMed1993

Processive interfacial catalysis by mammalian 85-kilodalton phospholipase A2 enzymes on product-containing vesicles: application to the determination of substrate preferences.

Substrate specificities of the human and rat kidney 85-kDa phospholipase A2 enzymes (hmw-PLA2) have been determined under conditions in which hydrolysis of substrate vesicles occurs without the desorption of enzyme from the interface (scooting mode catalysis). The rat kidney enzyme binds to vesicles of 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), which contain the substrate 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine (SAPC) and 10 mol% arachidonic acid (20:4) and 1-stearoyl-sn-glycero-3-phosphocholine (S-lyso-PC) as the hydrolysis reaction products, with a second-order rate constant k(on) approximately equal to 2 x 10(7) M-1 s-1. Upper limits of k(off) < or = 3 x 10(-4) s-1 and KD < = or 15 pM for the dissociation rate and equilibrium constants, respectively, are estimated from the vesicle binding measurements. The initial rates of hydrolysis of either radiolabeled 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphoserine (3H-SAPS), -phosphoethanolamine (3H-SAPE), -phosphoinositol (14C-SAPI), or -phosphate (3H-SAPA) and either 3H-SAPC or 14C-SAPC, which were incorporated into product-containing OPPC vesicles, were simultaneously measured with dual isotope radiometric assays. The plasmenylcholine 1-O-(Z-hexadec-1'-enyl)-2-arachidonyl-sn-glycero-3- phosphocholine (3H-PlasAPC) was also tested. Relative substrate specificity constants (Kcat/KM* values) were determined from the concentrations and initial rates of hydrolysis of the labeled substrates; the rank order of the values is SAPC approximately equal to SAPI approximately equal to PlasAPC > SAPE > SAPA approximately equal to SAPS. The maximal difference in specificity constants is 3.5-fold, indicating that the hmw-PLA2 does not significantly discriminate between phospholipids with different polar head groups. The diglyceride 1-stearoyl-2-arachidonyl-sn-glycerol is not a substrate for the human hmw-PLA2. Two mixtures of 1-stearoyl-2-acyl-sn-glycero-3-phosphocholine, which have different sn-2 acyl chains, were prepared and compared to SAPC as substrates. One mixture contained naturally-occurring unsaturated fatty acyl chains and the other contained a mixture of 20:4, all of its partially hydrogenated analogues (20:3, 20:2, and 20:1), and arachidic acid (20:0). The order of preference for the human hmw-PLA2 is sn-2-20:4 > sn-2-alpha-linolenoyl > sn-2-linoleoyl > sn-2-oleoyl > or = sn-2-palmitoyleoyl.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Purification of a mammalian protein geranylgeranyltransferase. Formation and catalytic properties of an enzyme-geranylgeranyl pyrophosphate complex.

A protein geranylgeranyltransferase (PGGT) that transfers the geranylgeranyl group from geranylgeranyl pyrophosphate (GGPP) to the cysteine residue in the C-terminal sequence Cys-Ali-Ali-Leu (Ali is an aliphatic amino acid) of proteins and peptides has been purified to apparent homogeneity from bovine brain. This was accomplished by affinity chromatography of partially purified enzyme on a gel containing a covalently attached hexapeptide SSCILL. This peptide was identified as a tight-binding ligand of the PGGT by employing a semi-random peptide synthetic strategy. The purified enzyme consists of two subunits of apparent molecular mass 40 and 48 kDa. Affinity-purified PGGT effectively catalyzes the prenylation of peptides that contain a C-terminal Leu or Phe residue. The PGGT forms a stable binary complex with intact GGPP that can be isolated by gel filtration. Addition of a peptide substrate to this complex results in the quantitative transfer of the prenyl group to the peptide. This transfer occurs without the equilibration of enzyme-bound GGPP with free GGPP. When the PGGT was incubated with farnesyl pyrophosphate, the amount of binary complex formed was about 25% of that formed with GGPP.

Alkyl and Aryl Transferases↗
PubMed1993

Human nonpancreatic secreted phospholipase A2: interfacial parameters, substrate specificities, and competitive inhibitors.

The rate and equilibrium parameters for the interfacial catalysis by recombinant human nonpancreatic secreted phospholipase A2 were determined. Results show that the enzyme binds to anionic interfaces with considerably higher affinity than to zwitterionic interfaces. The extent of hydrolysis per enzyme on anionic vesicles in the processive scooting mode shows that the enzyme is fully catalytically active as a monomer. Among several secreted phospholipases A2 tested, the human nonpancreatic secreted enzyme is unique in its ability to undergo slow intervesicle exchange either by dissociation from the interface followed by binding to a different vesicle or by promoting the fusion of vesicles. The equilibrium dissociation constants for calcium, substrate analogs, reaction products, and several competitive inhibitors bound to the enzyme at the interface were determined by monitoring the ligand-conferred protection of the active site histidine residue from alkylation by phenacyl bromide. The interfacial Michaelis-Menten parameters were determined from the analysis of the entire reaction progress curve and also by monitoring the effect of competitive inhibitors on the initial rate of hydrolysis in the scooting mode. The interfacial Michaelis constant (KM*) for the substrate 1,2-dimyristoylglycero-sn-3-phosphomethanol was determined to be considerably above the maximal attainable mole fraction of unity for the substrate in the bilayer. Substrate specificity studies show that the enzyme does not significantly discriminate between phospholipids that differ in the type of polar head group or in the degree of unsaturation of the fatty acyl chains. Competitive inhibitors are described that display a high degree of selectivity for binding to the nonpancreatic versus pancreatic phospholipase A2. The kinetic properties of the human nonpancreatic secreted phospholipase A2 suggest that the enzyme has evolved to hydrolyze substrates at anionic interfaces and at high calcium concentrations.

Catalysis↗
PubMed1992

High-level expression in Escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase A2.

Bee venom phospholipase A2 (BV-PLA2) is a hydrolytic enzyme that specifically cleaves the sn-2 acyl bond of phospholipids at the lipid/water interface. The same enzyme is also believed to be responsible for some systemic anaphylactic reactions in bee venom sensitized individuals. To study the structure/function relationships of this enzyme and to define the molecular determinants responsible for its allergenic potential, a synthetic gene encoding the mature form of BV-PLA2 was expressed in Escherichia coli. This enzyme was produced as a fusion protein with a 6xHis-tag on its amino-terminus yielding 40-50 mg of fusion protein per 1 of culture after metal ion affinity chromatography. A kallikrein protease recognition site was engineered between the 6xHis-tag and the amino-terminus of the enzyme allowing isolation of the protein with its correct N-terminus. Recombinant affinity purified BV-PLA2 was refolded, purified to homogeneity, and cleaved with kallikrein, resulting in a final yield of 8-9 mg of active enzyme per 1 of culture. The enzymatic and immunological properties of the recombinant BV-PLA2 are identical to enzyme isolated from bee venom indicating a native-like folding of the protein.

Amino Acid Sequence↗
PubMed1992

Interfacial catalysis by phospholipase A2: the rate-limiting step for enzymatic turnover.

The kinetics of the phospholipase A2-catalyzed hydrolysis of bilayer vesicles and mixed micelles of several oxyglycero and thioglycero analogues of phospholipids have been studied. The results with vesicles show that, depending on the source of the enzyme, the rates of hydrolysis of the oxy-containing long-chain phosphatidylmethanols are 2.5- to 28-fold higher compared to the rates of hydrolysis of the analogous thio substrates. The oxygen to sulfur substitution does not significantly alter the affinities of the enzymes for the reaction products or calcium. Since it is unlikely that sulfur substitution changes the rate constants for the formation and dissociation of the enzyme-product complex by the same factor, the element effects seen in the rates of hydrolysis of the oxy- and thioester phospholipids in vesicles are primarily due to a change in the rate constant for the chemical step of the catalytic turnover cycle. For bovine pancreatic phospholipase A2, various mutants with lower catalytic activity were used to show that the value of the element effect does not increase in the mutants. These results establish that, for the pancreatic phospholipase A2, the element effect is fully expressed, and the chemical step is fully rate-limiting for both oxyglycero and thioglycero phospholipids in vesicles. It was found that the element effect decreases from 7 to 1 when long-chain phosphatidylmethanols are present in micelles of a neutral diluent. This result suggests that the chemical step is not rate-limiting during the hydrolysis of these mixed micelle substrates.

Animals↗
PubMed1992

Kinetic analysis of a high molecular weight phospholipase A2 from rat kidney: divalent metal-dependent trapping of enzyme on product-containing vesicles.

The kinetics of hydrolysis of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine vesicles catalyzed by the high molecular weight phospholipase A2 from rat kidney show an anomalous behavior. The reaction progress lasts for several minutes and then stops after only 5-10% of the available substrate has been hydrolyzed. Addition of more enzyme but not more substrate leads to a new round of hydrolysis. Although this initially suggested that the enzyme becomes inactivated during the turnover, such a conclusion could not be substantiated. Addition of buffer containing 0.15 M NaCl and bovine serum albumin to the reaction after the progress ceased leads to the re-initiation of the lipolysis. The enzyme is not strongly inhibited by the reaction products. Although the enzyme does not bind irreversibly to vesicles composed of pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, it does become irreversibly trapped on vesicles that contain a critical mole percentage of reaction products. This trapping is the most likely explanation for the cessation of the reaction progress. Both the binding of enzyme to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles and the hydrolysis of 1-stearoyl-2-[3H]arachidonyl-sn-glycerophosphocholine contained in these vesicles require the presence of products. Furthermore, the trapping of enzyme is independent of catalytic turnover. The trapping is sensitive to the structure of the fatty acid present in the vesicles and requires the presence of divalent metals (either Ca2+, Sr2+, Ba2+, or Mg2+). Since the concentrations of the metals needed for the enzymatic activity correlate with the amounts needed to promote the trapping, it is suggested that the role of the metal is only to promote the interfacial binding of the enzyme.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Crystallographic analysis of transition state mimics bound to penicillopepsin: difluorostatine- and difluorostatone-containing peptides.

Difluorostatine- and difluorostatone-containing peptides have been evaluated as potent inhibitors of penicillopepsin, a member of the aspartic proteinase family of enzymes. Isovaleryl-Val-Val-StaF2NHCH3 [StaF2 = (S)-4-amino-2,2-difluoro-(R)-3-hydroxy-6-methylheptanoic acid] and isovaleryl-Val-Val-StoF2NHCH3 [StoF2 = (S)-4-amino-2,2-difluoro-3-oxo-6-methylheptanoic acid] have measured Ki's of 10 x 10(-9) and 1 x 10(-9) M, respectively, with this fungal proteinase. The StoF2-containing peptide binds 32-fold more tightly to the enzyme than the analogous peptide containing the non-fluorinated statine ethyl ester. Each compound was cocrystallized with penicillopepsin, intensity data were collected to 1.8-A resolution, and the atomic coordinates were refined to an R factor [formula: see text] of 0.131 for both complexes. The inhibitors bind in the active site of penicillopepsin in much the same fashion as do other statine-containing inhibitors of penicillopepsin analyzed earlier [James, M. N. G., Sielecki, A. Salituro, F., Rich, D. H., & Hofmann, T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6137-6141; James, M.N.G., Sielecki, A., & Hofmann, T. (1985) in Aspartic Proteinases and their Inhibitors (Kosta, V., Ed.) pp 163-177, Walter deGruyter, Berlin]. The (R)-3-hydroxyl group in StaF2 binds between the active site carboxyl groups of Asp33 and Asp213, making hydrogen-bonding contacts to each one. The ketone functional group of the StoF2 inhibitor is bound as a hydrated species, with the gem-diol situated between the two aspartic acid carboxyl groups in a manner similar to that predicted for the tetrahedral intermediate expected during the catalytic hydrolysis of a peptide bond [James, M. N. G., & Sielecki, A. (1985) Biochemistry 24, 3701-3713]. One hydrogen-bonding interaction from the "outer" hydroxyl group is made to O delta 1 of Asp33, and the "inner" hydroxyl group forms two hydrogen-bonding contacts, one to each of the carboxyl groups of Asp33 (O delta 2) and Asp213 (O delta 2). The only structural difference between the StaF2 and StoF2 inhibitors that accounts for the factor of 10 in their Ki's is the additional (R)-3-OH group on the tetrahedral sp3 carbon atom of the hydrated StoF2 inhibitor. The intermolecular interactions involving the fluorine atoms of each inhibitor are normal van der Waals contacts to one of the carboxyl oxygen atoms of Asp213 (F2-O delta 2 Asp213, 2.9 A). The observed stereochemistry of the bound StoF2 group in the active site of penicillopepsin has stimulated our reappraisal of the catalytic pathway for the aspartic proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)

Aspartic Acid Endopeptidases↗