Acyl-enzyme Species Research Articles

Formation of the Covalent Chymotrypsin·Antichymotrypsin Complex Involves No Large-scale Movement of the Enzyme

alpha(1)-Antichymotrypsin is a member of the serine proteinase inhibitor, or serpin, family that typically forms very long-lived, enzymatically inactive 1:1 complexes (denoted E*I*) with its target proteinases. Serpins share a conserved tertiary structure, in which an exposed region of amino acid residues (called the reactive center loop or RCL) acts as bait for a target proteinase. Within E*I*, the two proteins are linked covalently as a result of nucleophilic attack by Ser(195) of the serine proteinase on the P1 residue within the RCL of the serpin. This species is formally similar to the acyl enzyme species normally seen as an intermediate in serpin proteinase catalysis. However, its subsequent hydrolysis is extremely slow as a result of structural changes within the enzyme leading to distortion of the active site. There is at present an ongoing debate concerning the structure of the E*I* complex; in particular, as to whether the enzyme, bound to P1, maintains its original position at the top of the serpin molecule or instead translocates across the entire length of the serpin, with concomitant insertion of RCL residues P1-P14 within beta-sheet A and a large separation of the enzyme and RCL residue P1'. We report time-resolved fluorescence energy transfer and rapid mixing/quench studies that support the former model. Our results indicate that the distance between residue P1' in alpha(1)-antichymotrypsin and the amino terminus of chymotrypsin actually decreases on conversion of the encounter complex E.I to E*I*. These results led us to formulate a comprehensive mechanism that accounted both for our results and for those of others supporting the two different E*I* structures. In this mechanism, partial insertion of the RCL, with no large perturbation of the P1' enzyme distance, is followed by covalent acyl enzyme formation. Full insertion can subsequently take place, in a reversible fashion, with the position of equilibrium between the partially and fully inserted complexes depending on the particular serpin-proteinase pair under consideration.

‘pH-jump’ crystallographic analyses of γ-lactam–porcine pancreatic elastase complexes

beta-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain gamma-lactam analogues of monocyclic beta-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrolysis. Crystallographic analysis at pH 5 of an acyl-enzyme complex formed with PPE and one of these inhibitors revealed the ester carbonyl located in the oxyanion hole in a similar conformation to that observed in the structure of a complex formed between a heptapeptide (beta-casomorphin-7) and PPE. Only weak electron density was observed for the His-57 side chain in its 'native' conformation. Instead, the His-57 side chain predominantly adopted a conformation rotated approx. 90 degrees from its normal position. PPE-gamma-lactam crystals were subjected to 'pH-jumps' by placing the crystals in a buffer of increased pH prior to freezing for data collection. The results indicate that the conformation of the gamma-lactam-derived acyl-enzyme species in the PPE active site is dependent on pH, a result having implications for the analysis of other serine protease-inhibitor structures at non-catalytic pH values. The results help to define the stereoelectronic relationship between the ester of the acyl-enzyme complex, the side chain of His-57 and the incoming nucleophile during the reversible (de)acylation steps, implying it is closely analogous to the hydrolytic deacylation step during catalytic peptide hydrolysis.

Inhibition of the Broad Spectrum Nonmetallocarbapenamase of Class A (NMC-A) β-Lactamase from Enterobacter cloacae by Monocyclic β-Lactams

beta-Lactamases hydrolyze beta-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are known, are the primary cause of resistance to beta-lactam antibiotics. The class A beta-lactamases form the largest group. A novel class A beta-lactamase, named the nonmetallocarbapenamase of class A (NMC-A) beta-lactamase, has been discovered recently that has a broad substrate profile that included carbapenem antibiotics. This is a serious development, since carbapenems have been relatively immune to the action of these resistance enzymes. Inhibitors for this enzyme are sought. We describe herein that a type of monobactam molecule of our design inactivates the NMC-A beta-lactamase rapidly, efficiently, and irreversibly. The mechanism of inactivation was investigated by solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 A resolution. The structure shed light on the nature of the fragmentation of the inhibitor on enzyme acylation and indicated that there are two acyl-enzyme species that account for enzyme inhibition. Each of these inhibited enzyme species is trapped in a distinct local energy minimum that does not predispose the inhibitor species for deacylation, accounting for the irreversible mode of enzyme inhibition. Molecular dynamics simulations provided evidence in favor of a dynamic motion for the acyl-enzyme species, which samples a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima. A discussion of the likelihood of such dynamic motion for turnover of substrates during the normal catalytic processes of the enzyme is presented.

Hydrogen Bonding and Attenuation of the Rate of Enzymic Catalysis

Hydrogen bonds between a small molecule and an enzyme can potentially contribute significantly to the stability of the complex. Such electrostatic interactions can also lower energy barriers for reactions by solvation of high-energy species. A novel type of inhibitor is described in this report, which was designed to take advantage of a hydrogen bond that it makes to the active-site histidine of chymotrypsin to attenuate its basicity. Substrates of chymotrypsin acylate the active-site serine (of the catalytic triad), and the acyl-enzyme intermediate undergoes deacylation in a second step of the catalytic turnover. The active-site histidine (of the catalytic triad) serves as the general base in both steps of the turnover process. Such attenuation of basicity by hydrogen bonding was expected to impair catalysis by the enzyme. Two molecules of this type were synthesized that are based on the structure of the chymotrypsin substrate Ac-l-Ala-l-Ala-Gly-l-Phe methyl ester. These were methyl (2R,3R)-5-(N-acetyl-l-alanyl-l-alanyl)amino-2-benzyl-3-hydroxylpentanoate (1) and methyl (2R,3S)-5-(N-acetyl-l-alanyl-l-alanyl)amino-2-benzyl-3-hydroxylpentanoate (2). Compound 1 acylated chymotrypsin, but the acyl-enzyme species resisted deacylation. On the other hand, compound 2 did not even have the ability to acylate the active-site serine. Molecular modeling supported the assertion that compound 1 makes a critical hydrogen bond to the active-site histidine at the acyl-enzyme stage, whereas compound 2 does so at the preacylation complex. The concepts described herein are of general interest and should find applications for inhibition of enzymes that employ general acid−base chemistry for their catalytic processes.

Nuances of Mechanisms and Their Implications for Evolution of the Versatile β-Lactamase Activity: From Biosynthetic Enzymes to Drug Resistance Factors

β-Lactamases of classes A and C are believed to have evolved from bacterial enzymes involved in biosynthesis of the peptidoglycan, the so-called penicillin-binding proteins. All these enzymes undergo acylation at an active-site serine by β-lactam antibiotics as a common feature. However, the fate of the acyl-enzyme species is different for β-lactamases and penicillin-binding proteins; deacylation is rapid for the former, whereas it is slow for the latter. It is believed that the acquisition of the ability to deacylate the acyl-enzyme intermediate led to the evolution of β-lactamase activity, which is indispensable for the survival of bacteria in the face of challenge by β-lactam antibiotics. The mechanisms of deacylation of acyl-enzyme intermediates for β-lactamases are examined as a means to investigate structural factors in evolutionary descendency of classes A and C of β-lactamases from penicillin-binding proteins. It is known that in class A β-lactamases the hydrolytic water approaches the acyl-enzyme...

Crystal Structure of 6α-(Hydroxymethyl)penicillanate Complexed to the TEM-1 β-Lactamase from Escherichia coli: Evidence on the Mechanism of Action of a Novel Inhibitor Designed by a Computer-Aided Process

The crystal structure of the complex of the TEM-1 β-lactamase from Escherichia coli inhibited by 6α-(hydroxymethyl)penicillanic acid (1) is reported herein. This is the first structure for an acyl-enzyme intermediate with a substrate reported for a native class A β-lactamase. This compound was designed and synthesized as a molecule that would acylate the active site of the enzyme, but would resist deacylation by virtue of the fact that its C6α hydroxymethyl moiety was expected to occupy the space near the hydrolytic water molecule (J. Am. Chem. Soc. 1995, 117, 11055). The crystal structure of the acyl-enzyme species is closely similar to one of the two energy-minimized acyl-enzyme models generated in the course of the design aspect of the work. The crystal structure provides evidence for a number of mechanistic features for the inhibition process and the ultimate recovery of the activity. Our results reported herein are consistent with the side-chain carboxylate of Glu-166 being the active-site basic func...

The reduction of propionic anhydride by aldehyde dehydrogenase-NADH mixtures AT pH 7.

Although the oxidation of aldehydes by aldehyde dehydrogenase is usually considered to be irreversible, Hart & Dickinson (1978) have shown that anhydrides of carboxylic acids could be reduced to aldehydes by enzyme.NADH mixtures. In a later study Motion et al (1988) demonstrated that acylation of aldehyde dehydrogenase.NADH complexes by acetic anhydride led to the production of acetaldehyde and NAD+ in stoichiometric amounts. It is clear from these results, and the fact that 80% of the initial NADH concentration can be oxidised by anhydrides at an almost constant rate, that there is no intrinsic thermodynamic barrier to the reverse reaction once the appropriate acyl enzyme species is formed. The mechanism for reduction of aldehydes is presumably the reverse of that for aldehyde oxidation (Scheme I).

Structure at the active site of an acylenzyme of alpha-chymotrypsin and implications for the catalytic mechanism. An electron nuclear double resonance study.

The structure of the acylenzyme intermediate in the hydrolysis of the specific spin-label ester substrate methyl N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carbonyl)-L-tryptophan ate and its fluoro analogs catalyzed by alpha-chymotrypsin (EC 3.4.21.1) has been determined by electron nuclear double resonance (ENDOR) and molecular modeling methods. By a combination of kinetic and cryoenzymological methods, we have established conditions to stabilize the spin-labeled acylenzyme reaction intermediate. Proton ENDOR features specific for the substrate were assigned on the basis of specific deuteration. From the observed ENDOR shifts for protons and fluorines that correspond to principal hyperfine coupling components, the dipolar hyperfine coupling contributions were calculated to estimate electron-nucleus distances. With these dipolar separations as constraints, conformations of the substrate both free in solution and in the active site of alpha-chymotrypsin were determined by molecular graphics analysis. Comparison of the conformation of the bound substrate to that of the free substrate showed that formation of the acylenzyme requires significant torsional alteration in substrate structure. The structural relationships between active-site residues and the substrate in its ENDOR-assigned conformation are examined with respect to the requirements of stereoelectronic rules for formation and breakdown of the acylenzyme species.

.beta.-Substituted .beta.-phenylpropionyl chymotrypsins. Structural and stereochemical features in stable acyl enzymes

In order to develop effective alternate substrate inhibitors for the serine protease, we have prepared a series of beta-substituted beta-phenylpropionic acid esters related to some systems known to form stable acyl enzymes with alpha-chymotrypsin. Some of these compounds were prepared in enantiomerically pure form by asymmetric synthesis. Acyl enzyme species were generated from chymotrypsin by reaction with the active esters, and the progress of deacylation was monitored by the proflavin displacement assay. In some cases, it was possible to distinguish two different deacylation rates that correspond to the two enantiomers. beta-Phenylpropionic acyl enzymes with beta-substituents that are nonpolar were not especially stable, but a number of the polar derivatives and particularly the acylamino derivatives showed slow rates of deacylation (kd less than 0.005 min-1), with three systems showing deacylation enantioselectivities in the range of 500-1500. These results are consistent with a model in which additional stabilization of the acyl enzyme and enantioselectivity in the deacylation process derives from an additional hydrogen bond between the acyl enzyme species (as an acceptor) and the enzyme (as a donor). A number of active site residues that might be involved in this hydrogen bond are discussed.

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase. The effect of pH on the aldehyde binding reactions and a re-examination of the problem of the site of proton release in the mechanism.

Initial-rate measurements and stopped-flow spectrophotometric experiments over a wide range of pH implicate an enzyme group of pKa approximately 6.6 affecting the aldehyde binding reactions. It is possible, though not proved, that the group involved is the cysteine residue involved in catalysis. Stopped-flow fluorescence studies show that a group of pKa greater than 8.5 facilitates hydrolysis of the NADH-containing acyl-enzyme species. The identity of this group is quite unknown. Studies with 4-nitrobenzaldehyde show that this substrate gives marked substrate inhibition at quite low (less than 20 microM) concentrations. The mechanism of catalysis seems to be the same as for propionaldehyde oxidation. It is argued that proton release occurs with both substrates on hydrolysis of the NADH-containing acyl-enzyme and not before hydride transfer, as has been previously suggested [Bennett, Buckley & Blackwell (1982) Biochemistry 21, 4407-4413].

Reaction of [formula omitted]-nitrophenyl trimethylacetate with yeast carboxypeptidase. Evidence for an acyl-enzyme intermediate

The kinetics of the hydrolysis of p -nitrophenyl trimethylacetate catalyzed by yeast carboxypeptidase have been measured under conditions of substrate in excess and indicate that the release of p -nitrophenol in two discrete stages can be observed. A fast release of p -nitrophenol in a concentration approximating that of the enzyme is seen initially, followed by a slow release, corresponding to the “turnover” reaction of the ester. These observations provide strong support for the postulation of a three step reaction sequence including the formation and decomposition of not only a Michaelis complex but also an acyl-enzyme species.