The general modifier ("allosteric") unireactant enzyme mechanism: redundant conditions for reduction of the steady state velocity equation to one that is first degree in substrate and effector

The general unireactant modifier mechanism in the absence of product can be described by the following linked reactions: E + S k1 in equilibrium k-1 ES k3----E + P; E + I k5 in equilibrium k-5 EI; EI + S k2 in equilibrium k-2 ESI k4----EI + P; and ES + I k6 in equilibrium k-6 ESI where S is a substrate and I is an effector. A full steady state treatment yields a velocity equation that is second degree in both [S] and [I]. Two different conditions (or assumptions) permit reduction of the velocity equation to one that is first degree in [S] and [I]. These are (a) that k-2k3 = k-1k4 (Frieden, C., J. Biol. Chem. 239, pp. 3522-3531, (1964)) and (b) that the I-binding reactions are at equilibrium (Reinhart, G. D., Arch. Biochem. Biophys. 224, pp. 389-401 (1983)). It is shown that each condition gives rise to the other (i.e., if the I-binding reactions are at equilibrium, then k-2k3 must equal k-1k4 and vice-versa). If one assumes equilibrium for the I-binding steps, the velocity equation derived by the method of Cha (J. Biol. Chem. 243, pp. 820-825 (1968)) is apparently second degree in [I] (Segel, I. H., Enzyme Kinetics, p. 838, Wiley-Interscience (1975)), but reduces to a first degree equation when the relationship derived by Frieden is inserted. If one starts by assuming a single equilibrium condition for I binding, e.g., k-5[EI] = k5[E][I] or k-6[ESI] = k6[ES][I], then a traditional algebraic manipulation of the remaining steady state equations provides first degree expressions for the concentrations of all enzyme species and also discloses the Frieden relationship.     

Kinetics and mechanism of the serine beta-lactamase catalyzed hydrolysis of depsipeptides

Steady-state kinetic parameters have been determined for the hydrolysis of a series of acyclic depsipeptides (ester analogues of acyl-D-alanyl-D-alanine peptides) catalyzed by representative class C (Enterobacter cloacae P99) and class A (Bacillus cereus I, TEM-2, and Staphylococcus aureus PC1) beta-lactamases. The best of these substrates, and the one most used in this work, was m-[[(phenylacetyl)-glycyl]oxy]benzoic acid, whose rates of cleavage could be followed spectrophotometrically. The P99 enzyme also catalyzed the methanolysis of these substrates in aqueous methanol solutions. Quantitative evaluation of the effects of methanol on the kinetics of the competing hydrolysis and methanolysis reactions, and on the product distribution, supports a reaction mechanism involving an acyl-enzyme intermediate whose formation is rate-determining under conditions of substrate saturation. Consideration of the variation of these kinetic parameters with the structure of the depsipeptides and comparison with the analogous parameters for bicyclic beta-lactam substrates suggest that a variety of substrate binding modes exist on this enzyme. The class A enzymes, B. cereus beta-lactamase I and the TEM-2 beta-lactamase, catalyze depsipeptide and benzylpenicillin hydrolyses but not methanolysis. The acyl-enzyme derived from both types of substrate is thus shielded from external nucleophiles; the shielding is therefore not an effect, direct or indirect, of the thiazolidinyl group in the penicilloyl-enzyme. The class A beta-lactamase of the PC1 plasmid of S. aureus is distinctly different from the above two representatives of that class, in that it does catalyze methanolysis of depsipeptides (but not of benzylpenicillin). The methanolysis kinetics suggest that deacylation is rate-determining at saturation, a conclusion supported by the demonstration of an intermediate during the hydrolysis of m-[[(phenylacetyl)glycyl]oxy]benzoate, subsequent to leaving-group departure. The beta-lactamases have thus been shown to catalyze the hydrolysis of specific depsipeptides with comparable facility to that demonstrated by D-alanyl-D-alanine carboxypeptidase/transpeptidases. The former enzymes, however, differ in being unable to cleave the analogous peptides.     

Halogenated protocatechuates as substrates for protocatechuate dioxygenase from Pseudomonas cepacia

Substrates containing electron-withdrawing groups were reacted with protocatechuate 3,4-dioxygenase and oxygen. Haloprotocatechuates (5-fluoro-, 5-chloro-, 5-bromo-, 2-chloro-, and 6-chloroprotocatechuates) are oxygenated by the enzyme at rates 28- to 3000-fold lower than that with the native substrate. These lower rates are due to both deactivation of substrate to O2 attack, and to the formation of abortive enzyme-substrate (ES) complexes. Such ES complexes with haloprotocatechuates are spectrally distinct from the normal ES complex. 6-Chloroprotocatechuate produces changes more like those due to protocatechuate. The abortive ES complexes, when rapidly mixed with oxygen, decay to free enzyme and product monophasically, and the dependence of the rates on O2 concentration shows that a rate-limiting step precedes reaction with O2. Thus these complexes are rather unreactive toward O2, and the rate-limiting step in oxygenation is their conversion to active complexes. In contrast, the reaction of O2 with the enzyme and 6-chloroprotocatechuate is biphasic, the first phase being dependent on O2 concentration (2 X 10(4) M-1 S-1) and the second not (7 S-1). The intermediate formed after the first phase strongly resembles the second intermediate seen in the reaction of enzyme with protocatechuate and O2 (Bull, C., Ballou, D. P., and Otsuka, S., (1981) J. Biol. Chem. 256, 12681-12686), implying that the electron-withdrawing effect of the chlorine slows the O2 addition step considerably while the conversion to the second intermediate is hardly affected. When the enzyme cycles through several turnovers with 6-chloroprotocatechuate, an enzyme species is formed that resembles the unreactive ES complexes seen with the other haloprotocatechuates, indicating that a small amount of the unreactive complex is in equilibrium with the reactive complex and that during successive turnovers the enzyme is slowly converted into the unreactive form. The formation of this form correlates with the observation that in assays the rate of product formation gradually decreases with time.     

PH-dependence of the steady-state rate of a two-step enzymic reaction.

1. The pH-dependence is considered of a reaction between E and S that proceeds through an intermediate ES under "Briggs-Haldane' conditions, i.e. there is a steady state in ES and [S]o greater than [E]T, where [S]o is the initial concentration of S and [E]T is the total concentration of all forms of E. Reactants and intermediates are assumed to interconvert in three protonic states (E equilibrium ES; EH equilibrium EHS; EH2 equilibrium EH2S), but only EHS provides products by an irreversible reaction whose rate constant is kcat. Protonations are assumed to be so fast that they are all at equilibrium. 2. The rate equation for this model is shown to be v = d[P]/dt = (kcat.[E]T[S]o/A)/[(KmBC/DA) + [S]o], where Km is the usual assembly of rate constants around EHS and A-D are functions of the form (1 + [H]/K1 + K2/[H]), in which K1 and K2 are: in A, the molecular ionization constants of ES; in B, the analogous constants of E; in C and D, apparent ionization constants composed of molecular ionization constants (of E or ES) and assemblies of rate constants. 3. As in earlier treatments of this type of reaction which involve either the assumption that the reactants and intermediate are in equilibrium or the assumption of Peller & Alberty [(1959) J. Am. Chem. Soc. 81, 5907-5914] that only EH and EHS interconvert directly, the pH-dependence of kcat. is determined only by A. 4. The pH-dependence of Km is determined in general by B-C/A-D, but when reactants and intermediate are in equilibrium, C identical to D and this expression simplifies to B/A. 5. The pH-dependence of kcat./Km, i.e. of the rate when [S]o less than Km, is not necessarily a simple bell-shaped curve characterized only by the ionization constants of B, but is a complex curve characterized by D/B-C. 6. Various situations are discussed in which the pH-dependence of kcat./Km is determined by assemblies simpler than D/B-C. The special situation in which a kcat./Km-pH profile provides the molecular pKa values of the intermediate ES complex is delineated.     

Hydrolysis of peptides by carboxypeptidase A: equilibrium trapping of the ES2 intermediate

The cobalt absorption and electron paramagnetic resonance (EPR) spectra of cobalt carboxypeptidase undergo unique variations on formation of catalytic peptide and ester intermediates as previously recorded in cryoenzymologic experiments employing rapid-scanning spectroscopy and cryotrapping [Geoghegan, K. F., Galdes, A., Martinelli, R. A., Holmquist, B., Auld, D.S., & Vallee, B. L. (1983) Biochemistry 22, 2255-2262]. We here describe a means of stabilizing these intermediates, which we have termed "equilibrium trapping". It allows peptide intermediates to be observed for longer periods (much greater than 1 min) at ambient as well as subzero temperatures. The reaction intermediate with the rapidly turned over peptide substrate Dns-Ala-Ala-Phe is trapped when the cobalt enzyme (greater than 10 microM) has catalyzed the attainment of chemical equilibrium between high concentrations of the hydrolysis products Dns-Ala-Ala, 10 mM, and L-phenylalanine, 50 mM, and the product of their coupling Dns-Ala-Ala-Phe. Under these conditions, Dns-Ala-Ala-Phe is present in the equilibrated substrate-product reaction mixture at a level that exceeds the one predicted on the basis of K'eq for hydrolysis of this substrate and is close to the enzyme concentration. Other pairs of peptide hydrolysis products yield similar results. Visible absorption and EPR spectra of the cobalt enzyme show that the synthesized peptide binds to the active site in the mode previously recognized as the ES2 catalytic intermediate in peptide hydrolysis. Equilibrium trapping of the ES2 intermediate allows analysis of its physicochemical properties by methods that could not be employed readily under cryoenzymological conditions, e.g., circular dichroic and magnetic circular dichroic spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the action of papain on fluorescent peptide substrates.

Kinetic measurements have been performed on the action of papain on mansyl-Gly-Val-Glu-Leu-Gly and on mansyl-Gly-Gly-Val-Glu-Leu-Gly, both of which are cleaved solely at the Glu-Leu bond under the conditions of our experiments. Stopped-flow experiments have shown that, under conditions of enzyme excess, the enhancement of the fluorescence of the mansyl group upon association of each of the oligopeptide substrates with papain is a biphasic process. A very rapid initial increase in fluorescence is followed by a slower first-order fluorescence enhancement. The observed rate constant for the latter process is greater with the mansyl pentapeptide than with the mansyl hexapeptide. A similar biphasic fluorescence change is seen upon the interaction of the mansyl peptides with mercuripapain, but the second step is much slower than in the case of the active enzyme. The rate of the second step in the association of active papain with the mansyl paptides shows saturation with increasing enzyme concentration, supporting the view that an initial enzyme-substrate complex (ES) is converted in a first-order process to the complex (ES) that undergoes cleavage to form products. The hydrolysis of the Glu-Leu bond is associated with a first-order decrease in fluorescence, as a consequence of the formation of the mansyl peptide product, which is bound less strongly than the substrate. The rate constant for this process is about 140 times greater with the mansyl hexapeptide than with the mansyl pentapeptide, thus giving further indication of the importance of secondary enzyme-substrate interactions in the efficiency of papain catalysis. For each of the two mansyl peptides, the values of the rate constants and the apparent Michaelis constants associated with the cleavage of the Glu-Leu bond, as determined by stopped-flow measurements under conditions of enzyme excess, were the same, within the precision of the data, as those estimated from experiments under conditions of substrate excess, where the formation of Leu-Gly was determined by means of the fluorescamine reaction. This indicates that, with these substrates, the rate-limiting step in the overall catalytic process is associated with the breakdown of ES. Estimates are given of the dissociation constant of ES and of the rate constants in the interconversion of ES and ES.     

Reductive half-reaction of nitroalkane oxidase: effect of mutation of the active site aspartate to glutamate

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the DeltaG(++) for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.     

Fluoride effects along the reaction pathway of pyrophosphatase: evidence for a second enzyme.pyrophosphate intermediate

The fluoride ion is a potent and specific inhibitor of cytoplasmic pyrophosphatase (PPase). Fluoride action on yeast PPase during PP(i) hydrolysis involves rapid and slow phases, the latter being only slowly reversible [Smirnova, I. N., and Baykov, A. A. (1983) Biokhimiya 48, 1643-1653]. A similar behavior is observed during yeast PPase catalyzed PP(i) synthesis. The amount of enzyme.PP(i) complex formed from solution P(i) exhibits a rapid drop upon addition of fluoride, followed, at pH 7.2, by a slow increase to nearly 100% of the total enzyme. The slow reaction results in enzyme inactivation, which is not immediately reversed by dilution. These data show that fluoride binds to an enzyme.PP(i) intermediate during the slow phase and to an enzyme.P(i) intermediate during the rapid phase of the inhibition. In Escherichia coli PPase, the enzyme.PP(i) intermediate binds F(-) rapidly, explaining the lack of time dependence in the inhibition of this enzyme. The enzyme.PP(i) intermediate formed during PP(i) hydrolysis binds fluoride much faster (yeast PPase) or tighter (E. coli PPase) than the similar complex existing at equilibrium with P(i). It is concluded that PPase catalysis involves two enzyme.PP(i) intermediates, of which only one (immediately following PP(i) addition and predominating at acidic pH) can bind fluoride. Simulation experiments have indicated that interconversion of the enzyme.PP(i) intermediates is a partially rate-limiting step in the direction of hydrolysis and an exclusively rate-limiting step in the direction of synthesis.     

Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme.

J1/2 of the Tetrahymena ribozyme, a sequence of three A residues, connects the RNA-binding site to the catalytic core. Addition or deletion of bases from J1/2 improves turnover and substrate specificity in the site-specific endonuclease reaction catalyzed by this ribozyme: G2CCCUCUA5 (S) + G in-equilibrium G2CCCUCU (P) + GA5. These paradoxical enhancements are caused by decreased affinity of the ribozyme for S and P [Young, B., Herschlag, D., & Cech, T.R. (1991) Cell 67, 1007]. An additional property of these mutant ribozymes, decreased fidelity of RNA cleavage, is now analyzed. (Fidelity is the ability to cleave at the correct phosphodiester bond within a particular RNA substrate.) Introduction of deoxy residues to give "chimeric" ribo/deoxyribooligonucleotides changes the positions of incorrect cleavage. Previous work indicated that S is bound to the ribozyme by both base pairing and teritary interactions involving 2'-hydroxyl groups of S. The data herein strongly suggest that the P1 duplex, which consists of S base-paired with the 5' exon binding site of the ribozyme, can dock into tertiary interactions in different registers; different 2'-hydroxyl groups of S plug into tertiary contacts with the ribozyme in the different registers. It is concluded that the mutations decrease fidelity by increasing the probability of docking out of register relative to docking in the normal register, thereby giving cleavage at different positions along S. These data also show that the contribution of J1/2 to the teritiary interactions is indirect, not direct. Thus, a structural role of the nonconserved J1/2 is indicated: this sequence positions S to optimize tertiary binding interactions and to ensure cleavage at the phosphodiester bond corresponding to the 5' splice site. Substitution of sulfur for the nonbridging pro-RP oxygen atom at the normal cleavage site has no effect on (kcat/Km)S but decreases the fraction of cleavage at the normal site in reactions catalyzed by the -3A mutant ribozyme, which has all three A residues of J1/2 removed. Thus, the ribozyme chooses where to cleave S after rate-limiting binding of S, indicating that docking can change after binding and suggesting that the ribozyme could act processively. Indeed, it is shown that the +2A ribozyme cleaves at one position along an RNA substrate and then, before releasing that RNA product, cleaves it again.(ABSTRACT TRUNCATED AT 400 WORDS)     

Probing essential water in yeast pyrophosphatase by directed mutagenesis and fluoride inhibition measurements

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.     

N-(phenylacetyl)glycyl-D-aziridine-2-carboxylate, an acyclic amide substrate of beta-lactamases: importance of the shape of the substrate in beta-lactamase evolution

Certain acyclic depsipeptides, but not peptides, are substrates of typical beta-lactamases [Pratt, R.F., & Govardhan, C.P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1302]. This may reflect either the greater chemical reactivity of depsipeptides (and of beta-lactams, the natural substrates) than peptides or the greater ease of distortion of the depsipeptide (ester) than the peptide (amide) group into a penicillin-like conformation. The latter explanation has been shown to be more likely by employment of a novel beta-lactamase substrate. N-(phenylacetyl)glycyl-D-aziridine-2-carboxylate, which combines a high chemical reactivity with a close to tetrahedral amide nitrogen atom. Although this substrate was better (higher kcat/KM) than a comparable depsipeptide for beta-lactamases, it was poorer than the depsipeptide for the Streptomyces R61 D-alanyl-D-alanine peptidase (which catalyzes specific peptide hydrolysis). It therefore seems likely that one vital feature of the putative evolution of a DD-peptidase into a beta-lactamase would have been modification of the active site to, on one hand, accommodate bicyclic beta-lactams and, on the other, exclude productive binding of planar acyclic amides. Certain serine beta-lactamases and the R61 DD-peptidase also catalyze methanolysis and aminolysis by D-phenylalanine of the N-acylaziridine. The latter reaction, the first amide aminolysis shown to be catalyzed by a beta-lactamase, is a very close analogue of the transpeptidase reaction of DD-peptidases. The methanolysis reaction appeared to proceed by way of the same acyl-enzyme intermediate as formed from depsipeptides possessing the same acyl moiety as the aziridine. The kinetics of methanolysis were employed to determine whether acylation or deacylation was rate limiting to the hydrolysis reaction under saturating substrate concentrations. The kinetics of the aminolysis reaction, catalyzed by the Enterobacter cloacae P99 beta-lactamase, showed the characteristics of, and were interpreted in terms of, a sequential mechanism previously deduced for depsipeptides and this enzyme [Pazhanisamy, S., & Pratt, R. F. (1989) Biochemistry 28, 6875-6882]. This mechanism features two separate binding sites, only one of which is productive. Strikingly, the binding of the N-acylaziridine to the nonproductive site was very tight, such that essentially all hydrolysis at substrate concentrations above 0.1Km proceeded via the ternary complex; this could also be true of penicillins.     

Isolation and functional properties of an arginine-selective endoprotease from rat intestinal mucosa. A putative prosomatostatin convertase.

The endoproteolytic activity previously detected in rat intestinal mucosal extracts (Beinfeld M., Bourdais, J., Kuks, P., Morel, A., and Cohen, P. (1989) J. Biol. Chem. 264, 4460-4465), was purified to homogeneity as a 65-kDa molecular species. This putative proprotein-processing enzyme cleaves the peptide bond on the carboxyl side of a single arginine residue in hepta-[Leu62-Gln-Arg-Ser-Ala-Asn-Ser68] or trideca-[Asp56-Glu-Met-Arg-Leu-Glu-Leu-Gln-Arg-Ser-Ala-Asn-+ ++Ser68] peptides, reproducing the prosomatostatin sequence around Arg64, the locus for endoproteolytic release of either somatostatin-28 or its NH2-terminal fragment, somatostatin-28-(1-12), from their common precursor. This enzyme exhibits a strict selectivity for arginyl residues, as demonstrated with related substrates, and did not cleave at lysyl residues. Moreover, only arginyl residues belonging to peptides of the prosomatostatin family were cleaved, since no hydrolysis of peptides from other prohormones was detected. In addition, the arginine residue situated at position -5 on the NH2-terminal side of Arg64 not only did not function as a cleavage locus, but had no effect on the overall cleavage kinetics of the prosomatostatin-(56-68) peptide substrate. This enzyme also cleaved, but with much less efficiency, the peptide bond on the carboxyl side of an arginine in peptides containing either an Arg-Lys or a Lys-Arg doublet corresponding to prohormone cleavage sites. This enzyme was insensitive to divalent cation chelators, was completely inhibited by aprotinin and leupeptin, and was somewhat inhibited by other serine-protease inhibitors. It is concluded that this endoprotease is a serine protease and could be involved in prohormone or proprotein post-translational processing at single arginine cleavage sites.     

Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).     

Specificity and inhibition of proteases from human immunodeficiency viruses 1 and 2

Highly purified, recombinant preparations of the virally encoded proteases from human immunodeficiency viruses (HIV) 1 and 2 have been compared relative to 1) their specificities toward non-viral protein and synthetic peptide substrates, and 2) their inhibition by several P1-P1' pseudodipeptidyl-modified substrate analogs. Hydrolysis of the Leu-Leu and Leu-Ala bonds in the Pseudomonas exotoxin derivative, Lys-PE40, is qualitatively the same for HIV-2 protease as published earlier for the HIV-1 enzyme (Tomasselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., FitzGerald, D. J., Chaudhary, V. K., Pastan, I., and Heinrikson, R. L. (1990) J. Biol. Chem. 265, 408-413). However, the rates of cleavage at these two sites are reversed for the HIV-2 protease which prefers the Leu-Ala bond. The kinetics of hydrolysis of this protein substrate by both enzymes are mirrored by those obtained from cleavage of model peptides. Hydrolysis by the two proteases of other synthetic peptides modeled after processing sites in HIV-1 and HIV-2 gag polyproteins and selected analogs thereof demonstrated differences, as well as similarities, in selectivity. For example, while the two proteases were nearly identical in their rates of cleavage of the Tyr-Pro bond in the HIV-1 gag fragment, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, the HIV-1 protease showed a 64-fold enhancement over the HIV-2 enzyme in hydrolysis of a Tyr-Val bond in the same template. Accordingly, the HIV-2 protease appears to have a different specificity than the HIV-1 enzyme; it is better able to hydrolyze substrates with small amino acids in P1 and P1', but is variable in its rate of hydrolysis of peptides with bulky substituents in these positions. In addition to these comparisons of the two proteases with respect to substrate specificity, we present inhibitor structure-activity data for the HIV-2 protease. Relative to P1-P1' statine or Phe psi [CH2N]Pro-modified pseudopeptidyl inhibitors, compounds having Xaa psi[CH(OH)CH2]Yaa inserts were found to show significantly higher affinities to both enzymes, generally binding from 10 to 100 times stronger to HIV-1 protease than to the HIV-2 enzyme. Molecular modeling comparisons based upon the sequence homology of the two enzymes and x-ray crystal structures of HIV-1 protease suggest that most of the nonconservative amino acid replacements occur in regions well outside the catalytic cleft, while only subtle structural differences exist within the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Catalytic reaction profile for alcohol oxidation by galactose oxidase

Galactose oxidase is a remarkable enzyme containing a metalloradical redox cofactor capable of oxidizing a variety of primary alcohols during enzyme turnover. Recent studies using 1-O-methyl alpha-D-galactopyranoside have revealed an unusually large kinetic isotope effect (KIE) for oxidation of the alpha-deuterated alcohol (kH/kD = 22), demonstrating that cleavage of the 6,6'-di[2H]hydroxymethylene C-H bond is fully rate-limiting for oxidation of the canonical substrate. This step is believed to involve hydrogen atom transfer to the tyrosyl phenoxyl in a radical redox mechanism for catalysis [Whittaker, M. M., Ballou, D. P., and Whittaker, J. W. (1998) Biochemistry 37, 8426-8436]. In the work presented here, the enzyme's unusually broad substrate specificity has allowed us to extend these investigations to a homologous series of benzyl alcohol derivatives, in which remote (meta or para) substituents are used to systematically perturb the properties of the hydroxyl group undergoing oxidation. Quantitative structure-activity relationship (QSAR) correlations over the steady state rate data reveal a shift in the character of the transition state for substrate oxidation over this series, reflected in a change in the magnitude of the observed KIE for these reactions. The observed KIE values have been shown to obey a log-linear correlation over the substituent parameter, Hammett sigma. For the relatively difficult to oxidize nitro derivative, the KIE is large (kH/kD = 12.3), implying rate-limiting C-H bond cleavage for the oxidation reaction. This contribution becomes less important for more easily oxidized substrates (e.g., methoxy derivatives) where a much smaller KIE is observed (kH/kD = 3.6). Conversely, the solvent deuterium KIE is vanishingly small for 4-nitrobenzyl alcohol, but becomes significant for the 4-methoxy derivative (kH2O/kD2O = 1.2). These experiments have allowed us to develop a reaction profile for substrate oxidation by galactose oxidase, consisting of three components (hydroxylic proton transfer, electron transfer, and hydrogen atom transfer) comprising a single-step proton-coupled electron transfer mechanism. Each component exhibits a distinct substituent and isotope sensitivity, allowing them to be identified kinetically. The proton transfer component is unique in being sensitive to the isotopic character of the solvent (H2O or D2O), while hydrogen atom transfer (C-H bond cleavage) is independent of solvent composition but is sensitive to substrate labeling. In contrast, electron transfer processes will in general be less sensitive to isotopic substitution. Our results support a mechanism in which initial proton abstraction from a coordinated substrate activates the alcohol toward inner sphere electron transfer to the Cu(II) metal center in an unfavorable redox equilibrium, forming an alkoxy radical which undergoes hydrogen atom abstraction by the tyrosine-cysteine phenoxyl free radical ligand to form the product aldehyde.     

The cleavage step of ribonuclease P catalysis is determined by ribozyme-substrate interactions both distal and proximal to the cleavage site.

The cleavage step of bacterial RNase P catalysis involves concentration-independent processes after the formation of the ribozyme-substrate complex that result in the breaking of a phosphodiester bond. The 2'OH group at the cleavage site of a pre-tRNA substrate is an important determinant in the cleavage step. We determined here that in contrast to a tRNA substrate, the 2'OH at the cleavage site of two in vitro selected substrates has no effect, whereas a 2'OH located adjacent to the cleavage site has a similarly large effect on the cleavage step. This result indicates that a unique 2'OH in the vicinity of the cleavage site interacts with the ribozyme to achieve the maximal efficiency of the cleavage step. Individual modifications in a pre-tRNA substrate that disrupt ES interactions proximal to the cleavage site generally have little effect on the usage of this unique 2'OH. Ribozyme modifications that delete the interactions involving the T stem-loop of the tRNA have a large effect on the usage of this unique 2'OH and also alter the location of this 2'OH. We propose a new ES complex prior to the bond-breaking step in the reaction scheme to explain these results. This second ES complex is in fast equilibrium with the initial ES complex formed by bimolecular collision. The ribozyme interaction with this unique 2'OH shifts the equilibrium in favor of the second ES complex. The formation of the second ES complex may require optimal geometry of the two independently folding domains of this ribozyme to precisely position crucial functional groups and Mg2+ ions in the active site. Such a domain geometry is significantly favored by the RNase P protein. In the absence of the protein, spatial rearrangement of these domains in the ES complex may be necessary.     

The catalytic role of the copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase: a kinetic study of the H172A mutant

An essential histidine ligand to the electron transfer copper (CuH) of peptidylglycine alpha-hydroxylating monooxygenase (PHMcc) was mutated to an alanine and found to retain copper binding and hydroxylase activity [Jaron, S., et al. (2002) Biochemistry 41, 13274-13282]. An extensive kinetic and deuterium isotope effect study finds this mutant to maintain full coupling of O2 consumed to product formed despite a 3 order-of-magnitude decrease in kcat and a 300-fold decrease in kcat/Km(O2). Unexpectedly, electron transfer is not rate-limiting in H172A. Rather, the increased kinetic isotope effect (KIE) on kcat of 3.27 +/- 0.39 suggests that C-H bond cleavage has become more rate-limiting, implicating a role for His172 that goes beyond that of a simple ligand to CuH. The mechanistic implications are discussed.     

Energetics of enzyme catalysis. I. Isotopic experiments, enzyme interconversion, and oversaturation

An enzyme-catalyzed interconversion of one substrate, S, and one product P, by an enzyme that exists in two forms E1 and E2 where E1 binds S and E2 binds P, is considered S + E1 in equilibrium E1S in equilibrium E2P in equilibrium E2 + P. Under reversible conditions (where the concentrations of S and P are not far removed from their equilibrium values) it is shown that, in addition to the usual unsaturated and saturated behaviour there exists a third regime at high substrate concentration: the oversaturated region. In this region, the rate-limiting transition state is the interconversion of the unliganded forms of the enzyme: E1 and E2. Expressions for six different experiments involving deuterium, tritium and 14C labels are presented. By considering the results from these experiments, the nature and importance of the enzyme interconversion steps can be elucidated.     

Generation of adenosyl radical from S-adenosylmethionine (SAM) in biotin synthase

A mechanism of the C―S bond activation of S-adenosylmethionine (SAM) in biotin synthase is discussed from quantum mechanical/molecular mechanical (QM/MM) computations. The active site of the enzyme involves a [4Fe-4S] cluster, which is coordinated to the COO⁻ and NH₂ groups of the methionine moiety of SAM. The unpaired electrons on the iron atoms of the [4Fe-4S]²⁺ cluster are antiferromagnetically coupled, resulting in the S = 0 ground spin state. An electron is transferred from an electron donor to the [4Fe-4S]²⁺-SAM complex to produce the catalytically active [4Fe-4S]⁺ state. The SOMO of the [4Fe-4S]⁺-SAM complex is localized on the [4Fe-4S] moiety and the spin density of the [4Fe-4S] core is calculated to be 0.83. The C―S bond cleavage is associated with the electron transfer from the [4Fe-4S]⁺ cluster to the antibonding σ* C―S orbital. The electron donor and acceptor states are effectively coupled with each other at the transition state for the C―S bond cleavage. The activation barrier is calculated to be 16.0 kcal/mol at the QM (B3LYP/SV(P))/MM (CHARMm) level of theory and the C―S bond activation process is 17.4 kcal/mol exothermic, which is in good agreement with the experimental observation that the C―S bond is irreversibly cleaved in biotin synthase. The sulfur atom of the produced methionine molecule is unlikely to bind to an iron atom of the [4Fe-4S]²⁺ cluster after the C―S bond cleavage from the energetical and structural points of view.     

A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.

The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.