Azapeptides as inhibitors and active site titrants for cysteine proteinases

Ester and amide derivatives of alpha-azaglycine (carbazic acid, H2NNHCOOH), alpha-azaalanine, and alpha-azaphenylalanine (i.e., Ac-l-Phe-NHN(R)CO-X, where X = H, CH3, or CH2Ph, respectively) were synthesized and evaluated as inhibitors of the cysteine proteinases papain and cathepsin B. The ester derivatives inactivated papain and cathepsin B at rates which increased dramatically with leaving group hydrophobicity and electronegativity. For example, with 8 (R = H, X = OPh) the apparent second-order rate constant for papain inactivation was 67 600 M-1 s-1. Amide and P1-thioamide derivatives do not inactivate papain, nor are they substrates; instead they are weak competitive inhibitors (0.2 mM < Ki < 4 mM). Inactivation of papain involves carbamoylation of the enzyme, as demonstrated by electrospray mass spectrometry. Active site titration indicated a 1:1 stoichiometry for the inactivation of papain with 8, and both inactivated papain and cathepsin B are highly resistant to reactivation by dialysis (t1/2 > 24 h at 4 degrees C). Azaalanine derivatives Ac-L-Phe-NHN(CH3)CO-X inactivate papain ca. 400- 900-fold more slowly than their azaglycine analogues, consistent with the planar configuration at Nalpha of the P1 residue and the very substantial stereoselectivity of papain for L- vs D- residues at the P1 position of its substrates. Azaglycine derivative 9 (R = H, X = OC6H4NO2-p) inactivates papain extremely rapidly (>70 000 M-1 s-1), but it also decomposes rapidly in buffer with release of nitrophenol (kobs = 0.13 min-1); under the same conditions 8 shows <7% hydrolysis over 24 h. This nitrophenol release probably involves cyclization to an oxadiazolone since 17 (R = CH3, X = OC6H4NO2-p), which cannot form an isocyanate, releases nitrophenol almost as rapidly (kobs = 0.028 min-1). Cathepsin C, another cysteine proteinase with a rather different substrate specificity (i.e., aminopeptidase), was not inactivated by 8, indicating that the inactivation of papain and cathepsin B by azapeptide esters is a specific process. Their ease of synthesis coupled with good solution stability suggests that azapeptide esters may be useful as active site titrants of cysteine proteinases and probes of their biological function in vivo.     

Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein

Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of bacterial D-amino acid transaminase by beta-chloro-D-alanine

Purified D-amino acid transaminase from Bacillus sphaericus catalyzes an alpha,beta elimination from the D isomer of beta-chloroalanine to yield equivalent amounts of pyruvate, chloride, and ammonia; the L isomer of chloroalanine is not a substrate for this transaminase. During the beta elimination there is a synchronous loss in enzyme activity; the Kinact for beta-chloroalanine was estimated to be about 10 micrometers. The alpha-aminoacrylate-Schiff base intermediate formed after beta elimination of chloride ion is probably the key intermediate that partitions between one inactivation event for every 1500 turnovers. In the presence of D-alanine and alpha-ketoglutarate, which are good substrates for the transaminase activity of this enzyme, beta-chloroalanine is a potent, competitive inhibitor (K1 = 10 micrometers) with D-alanine and a weak, uncompetitive inhibitor with alpha-ketoglutarate.     

Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines

beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.     

Identification of the nonsubstrate steroid binding site of rat liver glutathione S-transferase, isozyme 1-1, by the steroid affinity label, 3beta-(iodoacetoxy)dehydroisoandrosterone

3beta-(Iodoacetoxy)dehydroisoandrosterone (3beta-IDA), an analogue of the electrophilic substrate, Delta5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3beta-IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3beta-IDA concentration, yielding an apparent Ki of 21 microM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17beta-estradiol-3,17-disulfate but, surprisingly, not by Delta5-androstene-3,17-dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme subunits indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the nonsubstrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.     

Tight binding of folate substrates and inhibitors to recombinant mouse glycinamide ribonucleotide formyltransferase

The binding of the prototypical folate inhibitor of de novo purine synthesis, 5,10-dideazatetrahydrofolate (DDATHF), and its hexaglutamate to recombinant trifunctional mouse glycinamide ribonucleotide formyltransferase (rmGARFT) was studied by equilibrium dialysis and by steady-state kinetics using sensitive assays that allowed initial rate calculations. rmGARFT was expressed in insect cells infected with a recombinant baculovirus and purified by a two-step procedure that allowed production of about 25 mg of pure protein/L of culture. The binding of DDATHF to GARFT was approximately 50-fold tighter than previously reported, with Kd and Ki values of 2-9 nM, making the parent form of this antifolate a tight-binding inhibitor. The binding of the hexaglutamate of DDATHF to rmGARFT had Kd and Ki values of 0.1-0.3 nM, consistent with the view that polyglutamation enhances binding of antifolates to GARFT. Kinetic analyses using either mono- or hexaglutamate substrate did not yield different values for the Ki for the hexaglutamate form of DDATHF, in contradiction with previous reports. Both the folate substrate commonly used to study GARFT, 10-formyl-5,8-dideazafolate, and its hexaglutamate were found to have very low Km values, namely, 75 and 7.4 nM, respectively, and the folate reaction products for these substrates were equally potent inhibitors, results which modify the interpretation of previous kinetic experiments. The product analog DDATHF and beta-glycinamide ribonucleotide bound to enzyme equally well in the presence and absence of the other, an observation at variance with the concept that GARFT obeys an ordered sequential binding of the substrates. We conclude that the kinetics of mouse GARFT are most consistent with a random order of substrate binding, that both the inhibitor DDATHF and the folate substrate are tight-binding ligands, and that polyglutamate forms enhance the affinity of both substrate and inhibitor by an order of magnitude.     

Interfacial catalysis by human 85 kDa cytosolic phospholipase A2 on anionic vesicles in the scooting mode

Analysis of phospholipases A2 on model phospholipid bilayers in which enzyme is essentially irreversibly bound at the lipid-water interface, termed "scooting mode", is a useful tool for studying the kinetic properties of interfacial enzymes. It is shown that human cytosolic 85 kDa phospholipase A2 (cPLA2) hydrolyzes sn-2-arachidonyl-containing phospholipids or the gamma-linolenoyl ester of 7-hydroxycoumarin (GLU) dispersed in vesicles of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (L-DOPM) in the scooting mode. Trapping of cPLA2 on L-DOPM vesicles is rapid and independent of product formation. Slowing of cPLA2-catalyzed hydrolysis of substrates present in phosphatidylmethanol and phosphatidylcholine vesicles is primarily due to apparent inactivation rather than to substrate depletion. cPLA2 phosphorylated on serine 505 by mitogen-activated protein kinase displays a 30% increase in the rate of sn-2-arachidonylphosphatidylcholine hydrolysis in the scooting mode compared to that of the nonphosphorylated enzyme. Kinetic parameters of cPLA2 acting on a variety of different phosphatidylmethanol vesicles were evaluated, and the results are discussed in terms of active site affinities for substrates and of lateral organization of substrates in the bilayer. A key result is that the sigmoidal kinetics reported previously using 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) vesicles are most prominent near the phase transition temperature of DMPM. No sigmoidal kinetics was observed using L-DOPM vesicles. The results of kinetic experiments and the behavior of a fluorescent substrate analog are consistent with nonideal mixing of substrate in DMPM vesicles, but not in L-DOPM vesicles, suggesting that apparent saturation and sigmoidal kinetics are more a result of nonideal mixing of substrate in DMPM vesicles than of active site binding of substrate. The fluorescence assay described using L-DOPM/GLU vesicles is useful for evaluating the interfacial behavior of cPLA2, including its substrate preferences and the effect of active site-directed inhibitors.     

Histidine decarboxylase of Lactobacillus 30a: inactivation and active-site labeling by L-histidine methyl ester

Histidine decarboxylase from Lactobacillus 30a is rapidly and irreversibly inactivated upon incubation with L-histidine methyl ester. The rate of inactivation is first-order with respect to remaining active enzyme and exhibits saturation kinetics with a kinact of 1.2 mM and an apparent first-order rate constant of 0.346 min-1 at pH 4.8 and 25 degrees C. On complete inactivation, 3 mol of [14C]histidine (from L-[14C]histidine methyl ester) and 2 mol of 14C (from L-histidine [14C]methyl ester) are bound in nondialyzable form per mol (190 000 g) of protein inactivated with a corresponding loss of three of the five DTNB-titratable--SH groups that are essential for activity of the native enzyme. Imidazole propionate, a competitive inhibitor of the enzyme, protects against inactivation, loss of --SH groups, and incorporation of radioactivity from both the histidine and the methyl ester moieties of the labeled inhibitor, and kinetic evidence indicates that imidazole propionate and histidine methyl ester compete for binding at the active site of histidine decarboxylase in a mutually exclusive manner. Treatment of the labeled protein with either alkali or hydroxylamine results in the quantitative release of radioactivity. These data suggest that inactivation of histidine decarboxylase by L-histidine methyl ester results from two different modes of interaction between the inhibitor and the active site of histidine decarboxylase; the major interaction involves an essential -SH group.     

Oxidative modification of aldose reductase induced by copper ion. Factors and conditions affecting the process

Bovine lens aldose reductase (ALR2) is inactivated by copper ion [Cu(II)] through an oxygen-independent oxidative modification process. A stoichiometry of 2 equiv of Cu(II)/enzyme mol is required to induce inactivation. While metal chelators such as EDTA or o-phenantroline prevent but do not reverse the ALR2 inactivation, DTT allows the enzyme activity to be rescued by inducing the recovery of the native enzyme form. The inactive enzyme form is characterized by the presence of 2 equiv of bound copper, at least one of which present as Cu(I), and by the presence of two lesser equivalents, with respect to the native enzyme, of reduced thiol residues. Data are presented which indicate that the Cu-induced protein modification responsible for the inactivation of ALR2 is the generation on the enzyme of an intramolecular disulfide bond. GSH significantly interferes with the Cu-dependent inactivation of ALR2 and induces, through its oxidation to GSSG, the generation of an enzyme form linked to a glutathionyl residue by a disulfide bond.     

Integral kinetics of multisubstrate enzyme reactions. Criteria of kinetic behavior and characteristic coordinates for solution of direct and reverse problems

General schemes of unbranched multisubstrate enzyme reactions associated with enzyme inactivation during catalysis are analyzed. Equations of integral kinetics at constant substrate concentrations and at depletion in one of the substrate are presented. Experimental dose-response curve characterized by additivity of some enzyme intermediates (absorption spectra, fluorescence, EPR, etc.) are theoretically analyzed. Also rapid equilibrium at certain stages of enzyme reaction is considered. The interrelationship of enzyme intermediates is a criterion of the kinetic behavior of enzyme reaction mechanism. New coordinates are suggested for the analysis of the integral kinetics of self-inactivating enzymes. Results of the analysis are used for interpretation of the data on arachidonic cascade enzymes.     

Anatomical variation of cerebral venous drainage: the theoretical effect on jugular bulb blood samples

A workshop was convened to discuss safety issues for traditional-approach HIV vaccines, especially inactivated vaccines. The topics included issues pertaining to (1) cell substrates used for production and (2) vaccine virus inactivation. The use of cell substrates such as tumor-derived continuous cell lines (TCLs) or virus-transformed. CLs may be the most feasible approach to provide commercial-scale virus yields. However, especially because of concerns about tumorigenicity, TCLs have not been used to produce preventive vaccines for human trials with healthy subjects in the United States. Residual TCL material (e.g., DNA, cellular proteins, viruses) may not be removed during purification of intact HIV virions to the same extent achievable for a recombinant protein. Manufacturing processes, e.g., physicochemical methods of destroying DNA, could decrease tumorigenicity risk. Methods to assess potential for tumorigenicity may need further development. Another potential substrate for viral production that merits further study is human peripheral blood mononuclear cells (PBMCs). Regardless of the cell substrate used, extensive testing for adventitious agents (including non-HIV retroviruses) is needed. Vaccine virus inactivation can be considered in statistical terms, i.e., the probability of a surviving infectious particle. One formula to determine a "safety margin" (SM) is reduction of titer in log10 for all inactivation steps minus initial viral infectivity in log10. Calculations for appropriate SMs should include all sources of variability (e.g., lot-to-lot differences). Ensuring a specified SM, as the lower bound of the 95% confidence interval, for production lots was discussed. Sensitivity and specificity of infectivity assays may present limitations.     

Identification of the subunit and important target peptides of pig heart NAD-dependent isocitrate dehydrogenase modified by the affinity label adenosine 5'-O-[S-(4-bromo-2, 3-dioxobutyl)thiophosphate]

Pig heart NAD-dependent isocitrate dehydrogenase is inactivated by adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPS-BDB) with incorporation of 1.78 mol of reagent/mol of average subunit. Complete protection against the inactivation is provided by 20 mM isocitrate + 1 mM Mn2+, and the incorporation is decreased to about 1.3 mol of reagent/mol of average subunit. The addition of NAD, NADH, or Mn2+ alone has little effect on the functional changes produced by AMPS-BDB, while ADP gives only partial protection against the inactivation. The ability of ADP to decrease the Km for isocitrate is not affected by the AMPS-BDB modification of the enzyme. These results indicate that the isocitrate substrate site is the target of AMPS-BDB. The enzyme has three types of subunits with a tetramer having the composition alpha2 beta gamma. Here, [2-3H]AMPS-BDB-modified subunits are separated by HPLC on a C4 reverse-phase column, after the treatment of the modified enzyme with 4 M urea. The predominant radioactivity is distributed in alpha and gamma subunits. However, evidence based on recombination of subunits from modified and unmodified enzymes indicates that only labeling of the alpha subunit is responsible for inactivation by AMPS-BDB. Subsequently, the separated modified subunits were chemically cleaved by CNBr and then purified by HPLC using a C18 column. The labeled peptides were further digested by pepsin, purified by HPLC, and sequenced. These results indicate that R88 and R98 from the alpha subunit are the major targets of AMPS-BDB which cause inactivation and that these are at or near the isocitrate site of the enzyme.     

The role of methionine-192 of the chymotrypsin active site in the binding and catalysis of mono(amino acid) and peptide substrates

We find that specific oxidation for the Met-192 residue in delta-chymotrypsin to methionine sulfoxide results in a twofold increase in Km(app) and unchanged kcat in the hydrolysis of N-acetyl mono(amino acid) amide substrates. However, the catalyzed hydrolyses of N-acetyl dipeptide amide substrates by (methionine sulfoxide)-192-delta-chymotrypsin (MS-delta-Cht) shows a four- to fivefold decrease in kcat and unchanged Km(app) with respect to delta-chymotrypsin. Hydrolysis of alpha-casein by MS-delta-Cht shows a similar 4.2-fold decrease in kcat. These results imply that the Met-192 acts differently with substrates that bind only in the primary, S1, binding site (i.e., AcPheNH2) from those that bind to more extended regions of the enzyme active site. In the binding of c+AcPheNH2 and AcTrpNH2, the results support a mechanism in which the Met-192 acts to slow the rate of sustrate dissociation from the Michaelis complex to free substrate and enzyme. This is in agreement with the x-ray crystallographic structure of dioxane inhibited alpha-chymotrypsin (Steitz, T., et al. (1969), J. Mol. Biol. 46, 337). However, this mechanism is not apparent when peptide and protein substrates bind. The decrease in kcat on Met-192 modification of approximately fivefold in the hydrolysis of polypeptide substrates show a small, but significant, catalytic contribution of the Met-192 toward the lowering of the energy of activation polypeptide substrate hydrolysis by chymotrypsin. This may support the crystallographic model of Fersht et al. (Fersht, A., et al. (1973), Biochemistry 12, 2035) in which it is proposed that the Met-192 participates in the distortion of bound polypeptide substrates toward the reaction transition-state configuration and, thus, plays a role in catalysis. However, if this mechanism occurs, the effect is small, only contributing about 1 kcal/mol to the lowering of the reaction activation energy.     

Active site specific inactivation of chymotrypsin by cyclohexyl isocyanate formed during degradation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea

Prolonged incubation of 1-(2-chloroethyl)-3-([1-14C]cyclohexyl)-1-nitrosourea with chymotrypsin resulted in covalent modification and concomitant inactivation of chymotrypsin via degradation of the nitrosourea to form cyclohexyl isocyanate. Cyclohexyl isocyanate was shown to be an active-site-specific inactivator of chymotrypsin. A cyclohexyl isocyanate to enzyme molar ratio of 0.63 was required to produce 50% enzyme inactivation, thus demonstrating the high specificity of inactivation. At 2.38 X 10(-4) M chymotrypsin this near stoichiometric inactivation was not significantly affected by the presence of 1, 5, and 10 mM L-lysine. Degradation of an excess of 1-(2-chloroethyl)-3-([1-14C]-cyclohexyl)-1-nitrosourea in the presence of enzyme yielded 1.11 +/- 0.07 mol of covalently bound [14C]cyclohexyl moiety per mol of enzyme inactivated. Short-term incubation demonstrated that the nitrosourea neither inhibited nor protected the enzyme from cyclohexyl isocyanate inactivation. Treatment of chymotrypsin with less than stoichiometric amounts of cyclohexyl isocyanate or titration of the active-site serine with phenylmethanesulfonyl fluoride followed by in situ degradation of excess 1-(2-chloroethyl)-3-([1-14C]cyclohexyl)-1-nitrosourea resulted in a decreased amount of covalently bound 14C proportional to the extent of inactivation by these reagents prior to 14C labeling. These results strongly suggest that cyclohexyl isocyanate, whether added directly or generated by CCNU degradation, reacted almost exclusively with the active site of the enzyme. The extent of this inactivation indicates that 70% of the CCNU degraded in such a manner as to form cyclohexyl isocyanate.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Pathway of activation by magnesium ions of substrates for the catalytic subunit of RNase P from Escherichia coli

The pathway is described for activation by Mg2+ of substrates for M1 RNA, the catalytic subunit of the RNase P from Escherichia coli. The dissociation constants are reported for binding of Mg2+ to the substrate and for the binding of the metal ion-substrate complex to the enzyme. The enzyme binds the substrate with the same affinity whether or not Mg2+ is already bound to the substate. However, only substrates with bound Mg2+ can make a productive ternary complex when combined with the enzyme. The presence of certain 2'-hydroxyl groups in the substrate is required to stabilize the binding of Mg2+ and, thereby, to increase the lifetime of the ternary complex. An energy profile for the reaction of M1 RNA with a small model substrate is presented and the role of Mg2+ bound to the substrate is discussed.     

Potent peptide inhibitors of human hepatitis C virus NS3 protease are obtained by optimizing the cleavage products

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.     

Inactivation of alpha-ketoglutarate dehydrogenase during oxidative decarboxylation of alpha-ketoadipic acid

alpha-Ketoglutarate dehydrogenase was inactivated irreversibly and completely during oxidation of alpha-ketoadipic acid. The inactivation was revealed both in the model system with ferricyanide and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither substrate depletion nor product accumulation induced the inactivation. The results obtained were compared with recent data on the enzyme inactivation during oxidation of alpha-ketoglutaric acid. The differences in the inactivation kinetics observed with the two substrates of the enzyme were analyzed. They seem not to reflect the different mechanisms of the inactivation, but, rather, depend on the changes in the rates of the individual stages of the process.     

Inactivation of bovine liver 2-keto-4-hydroxyglutarate aldolase by cyanide in the presence of aldehydes

Kinetic data show that the irreversible inactivation of liver 2-keto-4-hydroxyglutarate aldolase observed when the enzyme is incubated with an aldehydic substrate (or substrate analogue) in the presence of cyanide is a biphasic process and can, under certain conditions, involve a direct interaction between the enzyme and cyanide. The kinetic data are consistent with a scheme consisting of three competing reactions: (1) irreversible addition of cyanide to the enzyme-substrate Schiff base intermediate, (2) reversible cyanohydrin formation between cyanide and the aldehydic substrate (or substrate analogue), and (3) an interaction of cyanide with the enzyme which is not substrate dependent. Approximately 0.4 mol of cyanide is associated with 1 mol (120 000 g) of enzyme when 2-keto-4-hydroxyglutarate aldolase is incubated with [14-C]-cyanide followed by exhaustive dialysis; an ionic attachment possibly at a carboxylate binding site, is suggested. Whereas native enzyme, not treated with cyanide, has ten Nbs2-titratable sulfhydryl groups, approximately one less such group reacts with Nbs2 when the aldolase is incubated with cyanide (in the absence of aldehydic substrate). It is suggested that the binding of cyanide results in a conformational change of the enzyme; conformational changes in the presence of cyanide are confirmed by circular dichroism spectra.