Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Br?nsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.     

Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in chalcone isomerase

The reactivity of simple alkyl thiolates with N-ethylmaleimide (NEM) follows the Br?nsted equation, log kS- = log G + beta pK, with G = 790 M-1 min-1 and beta = 0.43. The rate constant for the reaction of the thiolate of 2-mercaptoethanol with NEM is 10(7) M-1 min-1, whereas the rate constant for the reaction of the protonated thiol is less than 0.0002 M-1 min-1. The intrinsic reactivity of the protonated thiol (SH) is over (5 X 10(10]-fold less than the thiolate (S-) and makes a negligible contribution to the reactivity of thiols toward NEM. The rate of NEM modification of chalcone isomerase was conveniently measured by following the concomitant loss in enzymatic activity. The pseudo-first-order rate constants for inactivation show a linear dependence on the concentration of NEM up to 200 mM and yield no evidence for noncovalent binding of NEM to the enzyme. Evidence is presented demonstrating that the modification of chalcone isomerase by NEM is limited to a single cysteine residue over a wide range of pH. Kinetic protection against inactivation and modification by NEM is provided by competitive inhibitors and supports the assignment of this cysteine residue to be at or near the active site of chalcone isomerase. The pH dependence of inactivation of the enzyme by NEM indicates a pK of 9.2 for the cysteine residue in chalcone isomerase. At high pH, the enzymatic thiolate is only (3 X 10(-5))-fold as reactive as a low molecular weight alkyl thiolate of the same pK, suggesting a large steric inhibition of reaction on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Systematic modulation of Michael-type reactivity of thiols through the use of charged amino acids.

A quantitative structure-reactivity relationship for the Michael-type addition of thiols onto acrylates was determined. Several thiol-containing peptides were investigated by examining the correlation between the second-order rate constant of their addition onto PEG-diacrylate and the pK(a) of the thiols within a peptide. By introducing charged amino acids in close proximity to a cysteine, the pK(a) of the thiol was systematically modulated by electrostatic interactions. Positive charges from the amino acid arginine decreased the pK(a) of the thiol and accelerated the reaction with acrylates while negative charges from aspartic acids showed the opposite effect. A linear correlation between thiolate concentrations and kinetic constants was found, confirming the role of thiolates as the reactive species in this Michael-type reaction. The relevant factors influencing the reactivity were the sign and the number of the neighboring charges, while the position of these charges had little effect on reactivity. These results provide a basis for the rational design of peptides, where the kinetics and thus selectivity of protein/peptide conjugation with polymeric structures via Michael-type addition reactions can be controlled.     

Kinetic studies of guinea pig liver transglutaminase reveal a general-base-catalyzed deacylation mechanism.

Guinea pig liver transglutaminase (TGase) reacts with 0.1 mM N-Cbz-L-Glu(gamma-p-nitrophenyl ester)Gly (5, prepared herein, K(M) = 0.02 mM) to undergo rapid acylation that can be followed spectrophotometrically at 400 nm (pH 7.0, 25 degrees C). Deacylation of the transiently formed thiolester acyl enzyme intermediate via catalytic aminolysis was studied in the presence of six primary amines of widely varying basicity (pK(NH+) = 5.6-10.5). Steady-state kinetic studies were performed to measure k(cat) and K(M) values for each amine substrate. A Br?nsted plot constructed through the correlation of log(k(cat)/K(M)) and pK(NH+) for each amine substrate displays a linear free-energy relationship with a slope beta(nuc) = -0.37 +/- 0.08. The shallow negative slope is consistent with a general-base-catalyzed deacylation mechanism in which a proton is removed from the amine substrate during its rate-limiting nucleophilic attack on the thiolester carbonyl. Kinetic isotope effects were measured for four acceptor substrates (water, kie = 1.1 +/- 0.1; aminoacetonitrile, kie = 5.9 +/- 1.2; glycine methyl ester, kie = 3.4 +/- 0.7; N-Ac-L-lysine methyl ester, kie = 1.1 +/- 0.1) and are consistent with a proton in flight at the rate-limiting transition state. The active site general-base implicated by these kinetic results is believed to be His-334, of the highly conserved TGase Cys-His-Asp catalytic triad.     

Formation and stability of the enolates of N-protonated proline methyl ester and proline zwitterion in aqueous solution: a nonenzymatic model for the first step in the racemization of proline catalyzed by proline racemase

Rate constants for the hydrolysis of L-proline methyl ester to form proline and methanol in D(2)O buffered at neutral pD and 25 degrees C and the deuterium enrichment of the proline product determined by electrospray ionization mass spectrometry are reported. The data give k(DO) = 5.3 +/- 0.5 M(-1) s(-1) as the second-order rate constant for carbon deprotonation of N-protonated proline methyl ester by deuterioxide ion in D(2)O at 25 degrees C and I = 1.0 (KCl). The data provide good estimates of carbon acidities of pK(a) = 21 for N-protonated proline methyl ester and pK(a) = 29 for proline zwitterion in water and of the second-order rate constant k(HO) = 4.5 x 10(-5) M(-1) s(-1) for carbon deprotonation of proline zwitterion by hydroxide ion at 25 degrees C. There is no detectable acceleration of the deprotonation of N-protonated proline methyl ester by the Br?nsted base 3-quinuclidinone in water, and it is not clear that such Br?nsted catalysis would make a significant contribution to the rate acceleration for deprotonation of bound proline at proline racemase. A comparison of the first-order rate constants k(HO)[HO(-)] = 4.5 x 10(-11) s(-1) for deprotonation of free proline zwitterion in water at pH 8 and k(cat) = 2600 s(-1) for deprotonation of proline bound to the active site of proline racemase at pH 8 shows that the enzymatic rate acceleration for proline racemase is ca. 10(13)-fold. This corresponds to a 19 kcal/mol stabilization of the transition state for deprotonation of the enzyme-bound carbon acid substrate by interaction with the protein catalyst. It is suggested that (1) much of the rate acceleration of the enzymatic over the nonenzymatic reaction in water may result from transfer of the substrate proline zwitterion from the polar solvent water to a nonpolar enzyme active site and (2) the use of thiol anions rather than oxygen anions as Br?nsted bases at this putative nonpolar enzyme active site may be favored, because of the smaller energetic price for desolvation of thiol anions than for desolvation of the more strongly solvated oxygen anions.     

Kinetic studies of rat kidney gamma-glutamyltranspeptidase deacylation reveal a general base-catalyzed mechanism

The enzyme gamma-glutamyltranspeptidase (GGT) is critical to cellular detoxification and leukotriene biosynthesis processes, as well as amino acid transport in kidneys. GGT has also been implicated in many important physiological disorders, including Parkinson's disease and inhibition of apoptosis. It binds glutathione as a donor substrate and initially forms a gamma-glutamyl-enzyme complex that can then react with a water molecule or an acceptor substrate (usually an amino acid or a dipeptide) to form glutamate or a product containing a new gamma-glutamyl-isopeptide bond, respectively, thus regenerating the free enzyme. Despite its important role in human physiology, the mechanisms of the reactions catalyzed by GGT are not well-known, particularly with respect to the deacylation step. We have synthesized a series of methionine amide derivatives whose alpha-ammonium groups have different pK(a) values. By using these compounds as acceptor substrates for GGT, we have constructed a Br?nsted plot and obtained a good correlation for log(k(norm)(cat,b)/K(b)) versus pK(a)(NH+) with a slope beta(nuc) of 0.84, consistent with a rate-limiting nucleophilic attack of the substrate amine on the acyl-enzyme intermediate. Isotope effect studies have shown that there is a proton in flight at the transition state, consistent with concerted deprotonation of the nucleophilic amine effected by an unidentified general base. A bell-shaped pH-rate profile has also been obtained for the deacylation step, reflecting the pK(a) values of the acceptor substrate (and/or that of a general base residue) and of a putative general acid that may be necessary for reprotonation of the active site nucleophile upon regeneration of the free enzyme. These data allow us to propose for the first time a detailed mechanism for this important step of the GGT-mediated reaction and to speculate about the origin of its acceptor substrate specificity.     

Catalytic mechanism of hamster arylamine N-acetyltransferase 2

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from AcCoA to primary arylamines, hydrazines, and hydrazides and play a very important role in the metabolism and bioactivation of drugs, carcinogens, and other xenobiotics. The reaction follows a ping-pong bi-bi mechanism. Structure analysis of bacterial NATs revealed a Cys-His-Asp catalytic triad that is strictly conserved in all known NATs. Previously, we have demonstrated by kinetic and isotope effect studies that acetylation of the hamster NAT2 is dependent on a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)) and not a general acid-base catalysis. In addition, we established that, after formation of the acetylated enzyme intermediate, the active-site imidazole, His-107, is likely deprotonated at physiological pH. In this paper, we report steady-state kinetic studies of NAT2 with two acetyl donors, acetyl coenzyme A (AcCoA) and p-nitrophenyl acetate (PNPA), and four arylamine substrates. The pH dependence of k(cat)/K(AcCoA) exhibited two inflection points at 5.32 +/- 0.13 and 8.48 +/- 0.24, respectively. The pK(a) at 5.32 is virtually identical with the previously reported pK(a) of 5.2 for enzyme acetylation, reaffirming that the first half of the reaction is catalyzed by a thiolate-imidazolium ion pair in the active site. The inflection point at 8.48 indicates that a pH-sensitive group on NAT2 is involved in AcCoA binding. A Br?nsted plot constructed by the correlation of log k(4) and log k(H)2(O) with the pK(a) for each arylamine substrate and water displays a linear free-energy relationship in the pK(a) range from -1.7 (H(2)O) to 4.67 (PABA), with a slope of beta(nuc) = 0.80 +/- 0.1. However, a further increase of the pK(a) from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k(4) value. Analysis of the pH-k(cat)/K(PABA) profile revealed a pK(a) of 5.52 +/- 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 +/- 0.04 on k(cat)/K(PABA). Normal solvent isotope effects of 4.8 +/- 0.1, 3.1 +/- 0.1, and 3.2 +/- 0.1 on the k(cat)/K(b) for anisidine, pABglu, and PNA, respectively, were also determined. These observations are consistent with a deacetylation mechanism dominated by nucleophilic attack of the thiol ester for arylamines with pK(a) values or=5.5. The general base is likely His-107 because the His-107 to Gln and Asn mutants were found to be devoid of catalytic activity. In contrast, an increase in pH-dependent hydrolysis of the acetylated enzyme was not observed over a pH range of 5.2-7.5. On the basis of these observations, a catalytic mechanism for the acetylation of arylamines by NAT2 is proposed.     

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Br?nsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.     

Detailed dissection of a new mechanism for glycoside cleavage: alpha-1,4-glucan lyase

The unusual enzyme, Gracilariopsis alpha-1,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of alpha-1,4-glucans via a beta-elimination reaction involving a covalent glycosyl-enzyme intermediate (Lee, S. S., Yu, S., and Withers, S. G. (2002) J. Am. Chem. Soc. 124, 4948-4949). The classical bell-shaped pH dependence of k(cat)/K(m) indicates two ionizable groups in the active site with apparent pK(a) values of 3.05 and 6.66. Br?nsted relationships of log k(cat) versus pK(a) and log(k(cat)/K(m)) versus pK(a) for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK(a) with low beta(lg) values of 0.32 and 0.33, respectively. The combination of these low beta(lg) values with large secondary deuterium kinetic isotope effects (k(H)/k(D) = 1.16 - 1.19) on the first step indicate a glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen at the transition state. Developed oxocarbenium ion character of the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin (K(i) = 20 and 130 nM, respectively) and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-alpha-D-glucopyranosyl fluoride, was the second elimination step shown to be rate-limiting. The large alpha-secondary deuterium kinetic isotope effect (k(H)/k(D) = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (k(H)/k(D) = 1.92) at C-2 confirm an E2 mechanism with strong E1 character for this second step. This considerable structural and mechanistic similarity with retaining alpha-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family.     

Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily

Thiols represent preferential targets of peroxynitrite in biological systems. In this work, we investigated the mechanisms and kinetics of the reaction of peroxynitrite with the dithiol dihydrolipoic acid (DHLA) and its oxidized form, lipoic acid (LA). Peroxynitrite reacted with DHLA being oxidation yields higher at alkaline pH. The stoichiometry for the reaction was two thiols oxidized per peroxynitrite. LA formation accounted for approximately 50% DHLA consumption at pH 7.4, probably reflecting secondary reactions between LA and peroxynitrite. Indeed, peroxynitrous acid reacted with LA with an apparent second-order rate constant (k(2app)) of 1400 M(-1) s(-1) at pH 7.4 and 37 degrees C. Nitrite and LA-thiosufinate were formed as reaction products. Surprisingly, the k(2app) for peroxynitrite-dependent DHLA oxidation was only 250 M(-1) s(-1) per thiol, at pH 7.4 and 37 degrees C. Testing various low-molecular-weight thiols, we found that an increase in the thiol pK (pK(SH)) value correlated with a decrease of k(2app) for the reaction with peroxynitrite at pH 7.4. The pK(SH) for DHLA is 10.7, in agreement with its modest reactivity with peroxynitrite.     

Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme

In the ribozyme from the hepatitis delta virus (HDV) genomic strand RNA, a cytosine side chain is proposed to facilitate proton transfer in the transition state of the reaction and, thus, act as a general acid-base catalyst. Mutation of this active-site cytosine (C75) reduced RNA cleavage rates by as much as one million-fold, but addition of exogenous cytosine and certain nucleobase or imidazole analogs can partially rescue activity in these mutants. However, pH-rate profiles for the rescued reactions were bell shaped, and only one leg of the pH-rate curve could be attributed to ionization of the exogenous nucleobase or buffer. When a second potential ionizable nucleobase (C41) was removed, one leg of the bell-shaped curve was eliminated in the chemical-rescue reaction. With this construct, the apparent pK(a) determined from the pH-rate profile correlated with the solution pK(a) of the buffer, and the contribution of the buffer to the rate enhancement could be directly evaluated in a free-energy or Br?nsted plot. The free-energy relationship between the acid dissociation constant of the buffer and the rate constant for cleavage (Br?nsted value, beta, = approximately 0.5) was consistent with a mechanism in which the buffer acted as a general acid-base catalyst. These data support the hypothesis that cytosine 75, in the intact ribozyme, acts as a general acid-base catalyst.     

Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase

l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.     

Oligopeptidase B: a new type of serine peptidase with a unique substrate-dependent temperature sensitivity

Oligopeptidase B, a member of the novel prolyl oligopeptidase family of serine peptidases, is involved in cell invasion by trypanosomes. The kinetic analysis of the reactions of oligopeptidase B, which preferentially cleaves peptides at two adjacent basic residues, has revealed significant differences from the trypsin-like serine peptidases. (i) The pH dependence of k(cat)/K(m) deviates from normal bell-shaped curves due to ionization of an enzymatic group characterized by a macroscopic pK(a) of approximately 8.3. The effect of this group is abolished at high ionic strength. (ii) The second-order acylation rate constants, k(cat)/K(m), are similar with the ester and the corresponding amide substrates, suggesting that their chemical reactivity does not prevail in the rate-limiting step. The kinetic deuterium isotope effects indicate that the rate-limiting step for k(cat)/K(m) is principally governed by conformational changes. (iii) The pH-k(cat)/K(m) profile and the very low rate constant for benzoyl-citrulline ethyl ester reveal a new kinetically influential group ionizing below the pK(a) of the active site histidine and indicate that the positive charge of arginine is essential for effective catalysis. (iv) The enzyme is inhibited by high concentrations of substrate. The mechanism of inhibition markedly varies with the reaction conditions. (v) The optimum temperature for the reactions of amide substrates is unusually low, slightly below 25 degrees C, whereas with benzoyl-arginine ethyl ester a linear Eyring plot is obtained up to 39 degrees C. The positive entropies of activation point to substantial reorganization of water molecules upon substrate binding.     

Ionization and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds of Bence Jones proteins and an Fab(t) fragment

The pK values and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds in Bence Jones proteins and the Fab(t) fragment of a myeloma protein (Jo) (IgGl, kappa) were determined by means of the reactions with chloroacetamide and DTNB, and of spectrophotometric titration. The two thiol groups of partially reduced type kappa Bence Jones protein dimers had the same pK values (pK = 9.76 at 0.2 ionic strength and 25 degrees C) and the same true second-order rate constants (k) toward chloroacetamide (k = 18.8 x 10(-2) M-1 . S-1). The two thiol groups of partially reduced type lambda Bence Jones protein dimers had different pK values but the variation of the pK values among the specimens was small (pK1 = 8.5-8.6 and pK2 = 9.5-9.7 at 0.2 ionic strength and 25 degrees C). The spectrophotometric titration of partially reduced Nag protein (type lambda) also showed that the two thiol groups have different pK values. The pK values of two thiol groups of the partially reduced Fab(t) fragment were determined as 8.51 and 9.76 at 0.2 ionic strength and 25 degrees C. The effect of ionic strength on the pK values of the thiol groups of partially reduced Nag protein and the pK values of the thiol groups in partially reduced Ta protein (type kappa) and in a hybrid molecule formed between partially reduced Ta protein and partially reduced and alkylated H chains indicated that the difference in pK values did not arise from electrostatic interaction between the two thiol groups, but that the pK values are intrinsically different. The true rate constants, k1 and k2, of the two thiol groups of type lambda Bence Jones proteins varied with the specimen (k1 = 1.9-5.7 x 10(-2) M-1 . S-1 and k2 = 18.5-25.0 x 10(-2) M-1 . S-1). The k1 and k2 values for Jo-Fab(t) were 7.21 x 10(-2) and 23.1 x 10(-2) M-1 . S-1, respectively. On the basis of these pK values and reactivities, we discuss the reformation of the interchain disulfide bonds from partially reduced Bence Jones proteins and immunoglobulins in the presence of oxidized glutathione.     

Mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase: kinetic studies

The catalytic mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) from family 39 of glycoside hydrolases has been subjected to a detailed kinetic investigation using a range of substrates. The enzyme exhibits a bell-shaped pH dependence of k(cat)/K(m), reflecting apparent pK(a) values of 4.1 and 6.8. The k(cat) and k(cat)/K(m) values for a series of aryl xylosides have been measured and used to construct two Br?nsted plots. The plot of log(k(cat)/K(m)) against the pK(a) of the leaving group reveals a significant correlation (beta(lg) = -0.97, r(2) = 0.94, n = 8), indicating that fission of the glycosidic bond is significantly advanced in the transition state leading to the formation of the xylosyl-enzyme intermediate. The large negative value of the slope indicates that there is relatively little proton donation to the glycosidic oxygen in the transition state. A biphasic, concave-downward plot of log(k(cat)) against pK(a) provides good evidence for a two-step double-displacement mechanism involving a glycosyl-enzyme intermediate. For activated leaving groups (pK(a) < 9), the breakdown of the xylosyl-enzyme intermediate is the rate-determining step, as indicated by the absence of any effect of the pK(a) of the leaving group on log(k(cat)) (beta(lg) approximately 0). However, a strong dependence of the first-order rate constant on the pK(a) value of relatively poor leaving groups (pK(a) > 9) suggests that the xylosylation step is rate-determining for these substrates. Support for the dexylosylation chemical step being rate-determining for activated substrates comes from nucleophilic competition experiments in which addition of dithiothreitol results in an increase in turnover rates. Normal secondary alpha-deuterium kinetic isotope effects ((alpha-D)(V) or (alpha-D)(V/K) = 1.08-1.10) for three different substrates of widely varying pK(a) value (5.15-9.95) have been measured and these reveal that the transition states leading to the formation and breakdown of the intermediate are similar and both steps involve rehybridization of C1 from sp(3) to sp(2). These results are consistent only with "exploded" transition states, in which the saccharide moiety bears considerable positive charge, and the intermediate is a covalent acylal-ester where C1 is sp(3) hybridized.     

Evidence that the active centre of chymopapain A is different from the active centres of some other cysteine proteinases and that the Br?nsted coefficient (beta nuc.) for the reactions of thiolate anions with 2,2'-dipyridyl disulphide may be decreased by reagent protonation.

The active centres of chymopapains A and B (jointly designated EC 3.4.22.6) and papaya (Carica papaya L.) peptidase A were investigated by using 2,2'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoic acid) as thiol-specific reactivity probes. Whereas the first active-centre pKa values for chymopapain B and papaya peptidase A are less than 5, is as the case for papain (EC 3.4.22.2) and ficin (EC 3.4.22.3), that for chymopapain A is about 6.8. The reason why the reactions of thiols of pKa approx. 6.5 with 2.2'-dipyridyl disulphide are essentially pH-independent in the pH range around the thiol pKa is delineated. The value of the Brønsted coefficient (beta nuc.) for the reactions of thiolate ions with the 2,2'-dipyridyl disulphide monocation appears to be smaller than its value for the corresponding reactions with the neutral disulphide.     

Limits of diffusion in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta.

The catalytic mechanism for the enzymatic hydrolysis of a series of paraoxon analogues by the phosphotriesterase from Pseudomonas diminuta has been determined. The Br?nsted plots relating the pKa of the leaving group to the observed kinetic parameters, Vmax and V/Km, are both nonlinear. This observation is consistent with a change in the rate-limiting step from chemical to physical events as the pKa of the leaving group is decreased. This conclusion is confirmed by the effects of solvent viscosity on Vmax and V/Km for the same series of analogues. The data were fitted to the scheme E k1A in equilibrium k2 EA k3----EP k7----E'P k9----E + products where EA is the enzyme-substrate complex, EP is the enzyme-product complex, E'P is the enzyme-product complex after a viscosity-independent unimolecular reaction, and the values for k1, k2, k7, and k9 are 4.1 X 10(7) M-1 s-1, 2550 s-1, 3370 s-1, and 5940 s-1, respectively. The magnitude of the chemical step, represented by k3, is dependent on the pKa of the leaving group phenol as predicted by the Br?nsted equation (log k3 = beta pKa + C) where beta = -1.8 and the constant (C) = 17.7. The magnitude of beta indicates that the transition state for substrate hydrolysis is very product-like.     

Factors that control the reactivity of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi

The amino acid sequences and X-ray structures of homodimeric triosephosphate isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side chain of the only interface cysteine (Cys14) of one subunit docks into loop 3 of the other subunit. This portion of the interface is also markedly similar in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of magnitude more susceptible to the thiol reagent methylmethane thiosulfonate (MMTS) than Cys14 of TbTIM. The causes of this difference were explored by measuring the second-order rate constant of inactivation by MMTS (k(2)) under various conditions. At pH 7.4, k(2) in TcTIM is 70 times higher than in TbTIM. The difference decreases to 30 when the amino acid sequence of loop 3 and adjoining residues of TbTIM are conferred to TcTIM (triple mutant). The pK(a) values of the thiol group of the interface cysteine of TcTIM and the triple mutant were 0.7 pH unit lower than in TbTIM. Because this difference could account for the different sensitivity of the enzymes to thiol reagents, we determined the k(2) of inactivation at equal levels of ionization of their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant to TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modify the pK(a) of the thiol group of the interface, albeit it diminished the rate of its derivatization by MMTS. In the presence of phosphoglycolate, under conditions in which the interface cysteines of the enzymes had equal levels of protonation, the difference in k(2) of TcTIM and TbTIM became smaller, whereas k(2) of the triple mutant was almost equal to that of TbTIM. Thus, from measurements of the reactivity of the interface cysteine in various conditions, it was possible to obtain information on the factors that control the dynamics of a portion of the dimer interface.     

Farnesylation of nonpeptidic thiol compounds by protein farnesyltransferase

Protein farnesyltransferase catalyzes the modification of protein substrates containing specific carboxyl-terminal Ca(1)a(2)X motifs with a 15-carbon farnesyl group. The thioether linkage is formed between the cysteine of the Ca(1)a(2)X motif and C1 of the farnesyl group. Protein substrate specificity is essential to the function of the enzyme and has been exploited to find enzyme-specific inhibitors for antitumor therapies. In this work, we investigate the thiol substrate specificity of protein farnesyltransferase by demonstrating that a variety of nonpeptidic thiol compounds, including glutathione and dithiothreitol, are substrates. However, the binding energy of these thiols is decreased 4-6 kcal/mol compared to a peptide derived from the carboxyl terminus of H-Ras. Furthermore, for these thiol substrates, both the farnesylation rate constant and the apparent magnesium affinity decrease significantly. Surprisingly, no correlation is observed between the pH-independent log(k(max)) and the thiol pK(a); model nucleophilic reactions of thiols display a Br?nsted correlation of approximately 0.4. These data demonstrate that zinc-sulfur coordination is a primary criterion for classification as a FTase substrate, but other interactions between the peptide and the FTase.isoprenoid complex provide significant enhancement of binding and catalysis. Finally, these results suggest that the mechanism of FTase provides in vivo selectivity for the farnesylation of protein substrates even in the presence of high concentrations of intracellular thiols.     

The thiol groups of the Folch-Pi protein from bovine white matter. Exposure, reactivity and significance.

The number and the reactivity of accessible thiol groups of the Folch-Pi apoprotein and proteolipid (50% of myelin proteins) were studied, by using a specific thiol-disulphide interchange reaction, in connection with the known solubility of this protein in organic and aqueous solvents. The high reactivity of 2,2'-dipyridyl disulphide towards thiol groups leads to the titration of 4.8 mol of SH groups/mol of protein (Mr 30000) in alkaline and acidic chloroform/methanol (2:1, v/v). Unlike previous findings, this value was consistently found from batch to batch and remained stable with time. In the proteolipid 1 mol of SH groups/mol was not accessible as compared with the apoprotein. In aqueous solvents, a similar number of 4.4 mol of SH groups/mol was also found. For the first time, kinetic studies carried out in chloroform/methanol discriminated between two classes of thiol groups. The reaction of 2 mol of SH groups/mol was characterized by apparent second-order rate constants whose values were 5-10-fold higher than those of the other class. Kinetic studies and cyanylation experiments in aqueous solvents also indicated the high reactivity of these thiol groups with Ellman's reagent. Together with kinetic results, studies on the stoichiometry of the interchange reaction of equimolar solutions of protein and disulphide indicate that these highly reactive thiol groups are near to each other in the amino acid sequence. The location of the thiol groups at the boundary between hydrophilic and hydrophobic domains of the Folch-Pi protein is suggested in connection with their possible structural and biological significance.