General Base Mechanism Research Articles

Stabilization of the imidazole ring of His-195 at the active site of chloramphenicol acetyltransferase

The imidazole of His-195 plays an essential role in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT). The structure of the binary complex of CATIII and chloramphenicol suggests that two unusual interactions might determine the conformation of the side chain of His-195: (i) an intraresidue hydrogen bond between its main chain carbonyl and the protonated N delta 1 of the imidazole ring and (ii) face-to-face van der Waals contact between the His-195 imidazole group and the aromatic side chain of Tyr-25. Tyr-25 also makes a hydrogen bond, via its phenolic hydroxyl, to the carbonyl oxygen of the substrate chloramphenicol. Replacement of Tyr-25 of CATIII by phenylalanine results in a modest increase in the Km for chloramphenicol (from 11.6 to 14.6 microM) and a 2-fold fall in kcat (599 to 258 s-1), indicative of a free energy contribution to transition state binding of 0.6 kcal mol-1 for the hydrogen bond between Tyr-25 and chloramphenicol. In contrast, substitution of Tyr-25 by alanine yields an enzyme that is dramatically impaired in its ability to bind chloramphenicol (Km = 173 microM). As kcat for Ala-25 CAT is also reduced (130 s-1), the loss of the aryl group results in a 69-fold decrease in kcat/Km, corresponding to a free energy contribution to binding and catalysis of 2.5 kcal mol-1. In addition to the loss of the hydrogen bond between Tyr-25 and chloramphenicol, the loss of substrate affinity in Ala-25 CAT may be a direct consequence of reduced hydrophobicity of the chloramphenicol-binding site and/or the loss of critical constraints on the precise conformation of the catalytic imidazole. However, as with wild type CAT, inactivation of Ala-25 CAT by the affinity reagent 3-(bromoacetyl) chloramphenicol is accompanied by modification solely at N epsilon 2 of His-195. Hence, the results demonstrate that tautomeric stabilization of the imidazole ring persists in the absence of van der Waals interactions with the side chain of Tyr-25, probably as a consequence of hydrogen bonding between the protonated N delta 1 and the carbonyl oxygen of His-195.

Crystal structure of the complex of carboxypeptidase A with a strongly bound phosphonate in a new crystalline form: comparison with structures of other complexes

O-[[(1R)-[[N-(Phenylmethoxycarbonyl)-L-alanyl]amino]ethyl] hydroxyphosphinyl]-L-3-phenyllacetate [ZAAP(O)F], an analogue of (benzyloxycarbonyl)-Ala-Ala-Phe or (benzyloxycarbonyl)-Ala-Ala-phenyllactate, binds to carboxypeptidase A with great affinity (Ki = 3 pM). Similar phosphonates have been shown to be transition-state analogues of the CPA-catalyzed hydrolysis [Hanson, J. E., Kaplan, A. P., & Bartlett, P. A. (1989) Biochemistry 28, 6294-6305]. In the present study, the structure of the complex of this phosphonate with carboxypeptidase A has been determined by X-ray crystallography to a resolution of 2.0 A. The complex crystallizes in the space group P2(1)2(1)2(1) with cell dimensions a = 61.9 A, b = 67.2 A, and c = 76.2 A. The structure of the complex was solved by molecular replacement. Refinement of the structure against 20,776 unique reflections between 10.0 and 2.0 A yields a crystallographic residual of 0.193, including 140 water molecules. The two phosphinyl oxygens of the inhibitor bind to the active-site zinc at 2.2 A on the electrophilic (Arg-127) side and 3.1 A on the nucleophilic (Glu-270) side. Various features of the binding mode of this phosphonate inhibitor are consistent with the hypothesis that carboxypeptidase A catalyzed hydrolysis proceeds through a general-base mechanism in which the carbonyl carbon of the substrate is attacked by Zn-hydroxyl (or Zn-water). An unexpected feature of the bound inhibitor, the cis carbamoyl ester bond at the benzyloxycarbonyl linkage to alanine, allows the benzyloxycarbonyl phenyl ring of the inhibitor to interact favorably with Tyr-198. This complex structure is compared with previous structures of carboxypeptidase A, including the complexes with the potato inhibitor, a hydrated keto methylene substrate analogue, and a phosphonamidate inhibitor. Comparisons are also made with the complexes of thermolysin with some phosphonamidate inhibitors.

The esterase-like activity of covalently bound human third complement protein

The acyl ester bond between the third complement protein, C3, and a variety of molcules is hydrolyzed spontaneously at neutral pH (Venkatesh et al., 1984). Modification of the free, single sulfhydryl group of bound C3 by thiol reagents suggested that a functional group other than the —SH acts as a “catalytic” group in this intramolecular hydrolytic reaction. Complete inhibition of the esterase-like activity is observed with stoichiometric amounts of mercuric chloride, palladium chloride, and the bifunctional organic mercurial, 3,6-bis-(acetoxymercuri)- o-toluidine [BAMT]. Since alkyl and aryl mercuric ions do not inhibit the esterase-like activity of C3-[ 3H]glycerol, it is conjectured that divalent mercury, palladium, and BAMT will form a complex with the —SH group and an atom of the “catalytic” group X having a lone pair of electrons. The structural features of C3 that are essential for the esterase-like activity remain intact after subjecting C3-[ 3H]glycerol to covalent chromatography on organomercurial agarose. Based on the observed effects of chemical reagents and the kinetic deuterium solvent isotope effect on the esterase-like activity, a general-base mechanism is proposed for the intramolecular hydrolysis of the acyl ester bond in covalently bound C3. The “catalytic” group X is located in the C3d region (residues 317–632 of the α chain), since C3d-[ 3H]glycerol also has esterase-like activity. A general-base mechanism mediated by the same “catalytic” group X may also apply to the formation of acyl ester bonds following the hydrolysis of the internal thiolester bond in native C3.

Chlorophyll [formula omitted] epimerization in organic solvents

Based on HPLC analyses of high resolution, the kinetic and mechanistic features of chlorophyll (Chl) a ⇄ a′ epimerization (reversible stereoisomerization on the cyclopentanone ring) have been studied for the first time in several organic solvents, and in diethyl ether containing a series of basic compounds. The epimerization-promoting effect of a base well parallels its p K a value, suggesting that epimerization proceeds via general base catalysis. Under favorable conditions the epimerization is completed within 1 min to give an equilibrium composition [ Chl a′] [ Chl a] ≈ 1 3 . With imidazole (Im) and its derivatives as bases, the epimerization rate constant is proportional to the squared base concentration, presumably due to the occurrence of a ‘tandem general base mechanism’ involving an Im dimer with enhanced basicity of one heteronitrogen atom. Chl a a′ epimerization is promoted also by histidine suspended in diethyl ether. These results are discussed referring to previous qualitative reports and to the epimeric composition of Chl-type pigments in photosynthetic apparatus.

On the Mechanism of Peptide Cleavage by Carboxypeptidase A and Related Enzymes

Abstract The evidence so far available indicates that carboxypeptidase A uses the general base mechanism for peptide hydrolysis. Based on model studies, the Glu-270 carboxylate probably performs three different proton transfers.

Rate and equilibrium studies of the reaction of oxyanions with 2-phenyloxazol-5(4H)-one

Equilibrium constants for the reaction of phenoxide ions with 2-phenyloxazol-5(4H)-one at 25 °C and 1 M ionic strength obey a Bronsted relationship (log kOH/kArO= log K′=–1.73pKArOH– 15.81) and are not subject to steric effects from ortho-substituents. Both forward and reverse rate parameters exhibit steric effects, and the Bronsted equations for meta- and para-substituted species are log kOH=–0.81 pKArOH+ 9.75, and log kArO= 0.95pKArOH– 6.40. There is no break in the Bronsted line in the region of pKArOH 5–11, consistent with a single transition-state. An upward deviation exists for oxyanions with low basicity (pKXOH < 5); one of these oxyanions, betaine, has a solvent deuterium oxide isotope effect for its reaction with the oxazolone greater than 2, consistent with a general base mechanism for attack for these species. The results for nucleophilic attack of phenolate anions are in agreement with a concerted displacement at the carbonyl group.

Stereochemical analysis of peptide bond hydrolysis catalyzed by the aspartic proteinase penicillopepsin.

The X-ray crystal structures of native penicillopepsin and of its complex with a synthetic analogue of the inhibitor pepstatin have been refined recently at 1.8-A resolution. These highly refined structures permit a detailed examination of peptide hydrolysis in the aspartic proteinases. Complexes of penicillopepsin with substrate and catalytic intermediates were modeled, by using computer graphics, with minimal perturbation of the observed inhibitor complex. A thallium ion binding experiment shows that the position of solvent molecule O39, between Asp-33(32) and Asp-213(215) in the native structure, is favorable for cations, a fact that places constraints on possible mechanisms. A mechanism for hydrolysis is proposed in which Asp-213(215) acts as an electrophile by protonating the carbonyl oxygen of the substrate, thereby polarizing the carbon-oxygen bond, a water molecule bound to Asp-33(32) (O284 in the native structure) attacks the carbonyl carbon as the nucleophile in a general-base mechanism, the newly pyramidal peptide nitrogen is protonated, either from the solvent after nitrogen inversion or by an internal proton transfer via Asp-213(215) from a hydroxyl of the tetrahedral carbon, and the tetrahedral intermediate breaks down in a manner consistent with the stereoelectronic hypothesis. The models permit the rationalization of observed subsite preferences for substrates and may be useful in predicting subsite preferences of other aspartic proteinases.

Evidence for the general base mechanism in carboxypeptidase A-catalyzed reactions: partitioning studies on nucleophiles and H2(18)O kinetic isotope effects.

Methanol does not detectably compete with water in carboxypeptidase-catalyzed cleavage of any substrate, although it is preferentially reactive in a model for the proposed nucleophilic mechanism for the enzyme that involves an anhydride intermediate. To test for such a common intermediate in the cleavage of related peptide and ester substrates, a method has been developed to examine H2(16)O-H2(18)O kinetic isotope-partitioning effects. The finding that benzoylglycylphenylalanine has an isotope effect of 1.019 +/- 0.002 while benzoylglycyl-beta-L-phenyl-lactate shows a small inverse isotope effect excludes most versions of a nucleophilic mechanism having a common anhydride intermediate. The bulk of the available evidence strongly favors the previously proposed general base mechanism.

The pyridine-catalysed reaction of methanol with phenyl isocyanate in tetrachloromethane

The title reaction is first order in catalyst and each reactant. Several 3- and 4-substituted pyridine catalysts generate a Bronsted plot with slope β of 0.49. This, the steric effect of 2,4,6-trimethylpyridine, the deuterium isotope effect, and the solvent effect are consistent with the general base mechanism of catalysis. The nucleophilic mechanism is considered relatively unlikely. 2-Pyridylmethanol reacts with phenyl isocyanate faster than does 4-pyridylmethanol, indicating intramolecular general base catalysis in the former case.

Reactions of 2-thiophenesulphonyl chloride with anion and neutral nucleophiles: Solvent effects on nucleophilic reactivity correlations

The reaction kinetics of 2-thiophenesulphonyl chloride with anion and neutral nucleophiles was studied in H 2O, D 2O and in protic solvents-H 2O (10% v v ) and aprotic solvents-H 2O (10% v v ) mixtures at 25°. Analysing the rate constants measured in water by Bronsted, Ritchie and Edwards equations the conclusion drawn that, for the nucleophilic order against the sulphonyl sulphur, basicity is of prime importance, although there may well be some dependence on polarizability and solvation. Solvent isotope effects show that the reactions occur by nucleophilic catalysis rather than by a general base mechanism. Water is the solvent in which there is the greater reactivity than in either protic solvents or aprotic-protic mixtures. By solubility measurements and applying Parker's equation the contributions of solvation energies of both reactants and transition states to the free energy of activation are calculated. Solvent effects on nucleophilic reactivities are discussed in terms of S parameters (similar to Ritchie N + parameters), and by the approach of multiparameter empirical correlations. The data point out that solvation plays a large role on nucleophilic order. A complete comprehension of the problem would require an equation that takes into some account solvent effects. The homogeneous comparison of 2-thiophenesulphonyl chloride data with those of α-disulphone, p-anisyl p-methoxybenzenesulphinyl sulphone and benzenesulphonyl chloride shows that the same factors are involved in driving the nucleophilic reactivity for these compounds.

Comparison of the structures of carboxypeptidase A and thermolysin.

Chemistry and Physics, University of Oregon, Eugene, Oregon 97403 Using the known three-dimensional structures of the zinc metallopeptidases carboxypeptidase A and thermolysin, the two enzymes are compared by superimposing one active site upon the other and identifying corresponding structural and functional elements. The overall folding of the respec- tive enzymes is quite different, but the active sites have several features in common, and the modes of binding of dipeptide inhibitors are very similar. The known differences in specificity of the two enzymes are readily explained in terms of the observed differences between the respective substrate binding sites. Also, it is clear that the similarities between carboxypeptidase A and thermolysin are due to convergent rather than divergent evolution. The close struc- tural correspondence in the immediate vicinity of the zinc suggests that these two neutral proteases from mammalian and bacterial sources may have similar mechanisms for peptide hydrolysis. For both thermolysin and carboxypepti- dase A, peptide substrates bind to the enzyme with the carbonyl oxygen of the scissile peptide displacing a bound water molecule and becoming the fourth zinc ligand. Evi- dence is presented that for both enzymes, hydrolysis pro- ceeds by a general base mechanism with Glu 143 of thermol- ysin, or Glu 270 of carboxypeptidase A, promoting the attack of a water molecule on the carbonyl carbon of the substrate. There is no tyrosine in the thermolysin active site which is analogous to Tyr 248 of carboxypeptidase A. In contrast, the imidazole of His 231 is the presumptive proton donor. The comparison of the active sites of thermol- ysin and carboxypeptidase A does not obviously confirm that in carboxypeptidase A Tyr 248 acts as a proton donor in peptide hydrolysis, but neither does it rule out this possibility.

Nucleophilic versus general base catalysis in phosphyl (PV) group transfer: application to α-chymotrypsin action

The hydrolysis of aryl dimethylphosphinates is shown to be catalysed via a nucleophilic pathway by a series of bases including imidazole which is less efficient than phosphate dianion. The more reactive nucleophilic pathway is allowed because dimethylphosphinate is less sterically hindered than diphenyl phosphinate where general base-catalysis predominates. Acylation of α-chymotrypsin by 4-nitrophenyl diphenylphosphinate, a bona fide general base mechanism, has a low solvent deuterium oxide isotope effect not characteristic of such a mechanism.

Substituent effects on the chymotrypsin-catalyzed hydrolysis of specific ester substrates.

The substituent effect on the chymotrypsin-catalyzed hydrolysis of several phenyl esters of specific substrates has been studied. The second-order acylation rate constants (kcat/Km(app)) obey a linear free energy relationship with ρ = +0.63 for phenyl hippurates and ρ = +0.46 for phenyl N-benzyloxycarbonyl-L-tryptophanates when substituents are introduced into the phenyl group of the ester function. These results further support the previously proposed general acid – general base mechanism for the acylation reaction and the formation of a tetrahedral intermediate in the course of the reaction.

Hydrolysis of phosphinic esters: general-base catalysis by imidazole

Imidazole catalyses the hydrolysis of aryl diphenylphosphinates (I). At 25° the Hammett-σ-ρ value for the bimolecular rate constant (kim) is 2·88 and at 55° is 2·64; hydroxide-ion catalysis (kOH) at 25° has a smaller ρ value 1·55. Ratio of phosphate dianion catalysis (kHPO4=) to kim is ca. 5 for 4-nitrophenyl diphenylphosphinate; the deuterium oxide solvent isotope effect on kim is 2·15 and no departure from 1:1 stoicheiometry was observed in the imidazole-catalysed hydrolysis of the 4-nitrophenyl ester. These results indicate that imidazole acts as a general-base catalyst. Nucleophilic catalysis is observed with imidazole-catalysed hydrolysis of 4-nitrophenyl dimethyl-phosphate, diphenylphosphate and di-4-nitrophenylphosphate (kimH/kimD= 1·03, 1·02, 1·53 respectively). Steric crowding of the transition state renders nucleophilic catalysis inefficient for the diphenylphosphinate esters.A diagnostic tool not previously employed to distinguish general-base from nucleophilic catalysis by imidazole is discussed. The good Hammett-σ plot for imidazole catalysis in diphenylphosphinate ester hydrolysis argues for a general-base mechanism and for a stepwise rather than a concerted displacement.

Steric effects in the nucleophilic versus general base-catalysed intramolecular carboxy-group-assisted solvolysis of esters

Intramolecular participation by carboxylate anion in the solvolysis of the hindered ester 2-carboxyphenyl mesitoate in anhydrous methanol, occurs entirely by a nucleophilic mechanism in contrast to aspirin which previously has been shown to involve the kinetically equivalent general base mechanism.

The complex active sites of bacterial neutral proteases in relation to their specificities

This chapter describes the activity and the specificity of thermolysin and related bacillus metallopeptidases. The pH-activity profiles of thermolysin and Thermolysin-Like Proteases (TLPs) follow a bell-shaped curve with maximal activity at or near pH 7.0 for both proteolysis. Mature thermolysin comprises 316 residues, forming a bilobal structure in which the N-terminal region is predominantly β-pleated sheet and the C-terminal domain is predominantly α-helical. The catalytically essential zinc atom is located in a deep active-site cleft that lies between the two lobes. The crystallographic studies on thermolysin, coupled with parallel studies on the zinc endopeptidase carboxypeptidase A, have led to a general base mechanism of action being proposed for the zinc peptidase family, with the glutamate of the HEALTH sequence polarizing the zinc-bound water molecule which subsequently attacks the scissile bond. Another histidine residue is proposed to contribute by stabilizing the transition state. Comparison of the crystal structures of thermolysin–ligand complexes with the structure of the free enzyme strongly indicates that thermolysin undergoes a conformational change upon substrate or inhibitor binding.