Kinetic characterization of the acyl-enzyme mechanism for beta-lactamase I.
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ABSTRACT:
SUBMITTER: Martin MT
PROVIDER: S-EPMC1135174 | biostudies-other | 1988 Sep
REPOSITORIES: biostudies-other
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Project description:The discovery that the mechanism of beta-lactam hydrolysis catalyzed by the class A (active site serine-dependent) beta-lactamases proceeds via an acyl-enzyme intermediate was made thirty years ago. Since this discovery, the active site circumstance that enables acylation of the active site serine and further enables hydrolytic deacylation of the acyl-serine intermediate, has received extraordinary scrutiny. The justification for this scrutiny is the direct relevance of the beta-lactamases to the manifestation of bacterial resistance to the beta-lactam antibiotics, and the subsequent (to the discovery of the beta-lactamase acyl-enzyme) recognition of the direct evolutionary relationship between the serine beta-lactamase acyl-enzyme, and the penicillin binding protein acyl-enzyme that is key to beta-lactam antibiotic activity. This short review describes the early events leading to the recognition that serine beta-lactamase catalysis proceeds via an acyl-enzyme intermediate, and summarizes several of the key mechanistic studies--including infrared spectroscopy, cryoenzymology, beta-lactam design, and x-ray crystallography--that have been exploited to understand this pivotal catalytic intermediate.
| S-EPMC6902449 | biostudies-literature
Project description:Methanol or ethanol can replace water in the action of certain chromosomal beta-lactamases on benzylpenicillin: the products are alpha-methyl or alpha-ethyl benzylpenicilloate. The beta-lactamases were from a mutant of Pseudomonas aeruginosa 18S that produces the enzyme constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol. 128, 155-159] and from Escherichia coli K12 (the ampC beta-lactamase) [Lindström, Boman & Steele (1970) J. Bacteriol. 101, 218-231]. The variation of the rates of alcoholysis and hydrolysis with concentration of alcohol show that the rate-determining step is breakdown of an intermediate. This intermediate is likely to be the acyl-enzyme. The esters, alpha-methyl or alpha-ethyl benzylpenicilloate, are themselves substrates for the Pseudomonas beta-lactamase, benzylpenicilloic acid being formed. Thus this beta-lactamase can be an esterase. The kinetics for the hydrolysis of cloxacillin by the Pseudomonas beta-lactamase are consistent with the acyl-enzyme, formed by acylation of serine-80, being an intermediate in the overall hydrolysis.
| S-EPMC1153862 | biostudies-other
Project description:Cryoenzymology techniques were used to facilitate trapping an acyl-enzyme intermediate in beta-lactamase I catalysis. The enzyme (from Bacillus cereus) was investigated in aqueous methanol cryosolvents over the 25 to -75 degrees C range, and was stable and functional in 70% (v/v) methanol at and below 0 degree C. The value of kcat. decreased linearly with increasing methanol concentration, suggesting that water is a reactant in the rate-determining step. In view of this, the lack of incorporation of methanol into the product means that the water molecule involved in the deacylation is shielded from bulk solvent in the enzyme-substrate complex. From the lack of adverse effects of methanol on the catalytic and structural properties of the enzyme we conclude that 70% methanol is a satisfactory cryosolvent system for beta-lactamase I. The acyl-enzyme intermediate from the reaction with 6-beta-(furylacryloyl)amidopenicillanic acid was accumulated in steady-state experiments at -40 degrees C and the reaction was quenched by lowering the pH to 2. H.p.l.c. experiments showed covalent attachment of the penicillin to the enzyme. Digestion by pepsin and trypsin yielded a single labelled peptide fragment; analysis of this peptide was consistent with Ser-70 as the site of attachment.
| S-EPMC1133516 | biostudies-other
Project description:The reactions between single crystals of the SHV-1 beta-lactamase enzyme and the carbapenems, meropenem, imipenem, and ertapenem, have been studied by Raman microscopy. Aided by quantum mechanical calculations, major populations of two acyl-enzyme species, a labile Delta (2)-pyrroline and a more tightly bound Delta (1)-pyrroline, have been identified for all three compounds. These isomers differ only in the position of the double bond about the carbapenem nucleus. This discovery is consonant with X-ray crystallographic findings that also identified two populations for meropenem bound in SHV-1: one with the acyl CO group in the oxyanion hole and the second with the acyl group rotated 180 degrees compared to its expected position [Nukaga, M., Bethel, C. R., Thomson, J. M., Hujer, A. M., Distler, A. M., Anderson, V. E., Knox, J. R., and Bonomo, R. A. (2008) J. Am. Chem. Soc. (in press)]. When crystals of the Delta (1)- and Delta (2)-containing acyl-enzymes were exposed to solutions with no carbapenem, rapid deacylation of the Delta (2) species was observed by kinetic Raman experiments. However, no change in the Delta (1) population was observed over 1 h, the effective lifetime of the crystal. These observations lead to the hypothesis that the stable Delta (1) species is due to the form seen by X-ray with the acyl carbonyl outside the oxyanion hole, while the Delta (2) species corresponds to the form with the carbonyl inside the oxyanion hole. Soak-in and soak-out Raman experiments also demonstrated that tautomeric exchange between the Delta (1) and Delta (2) forms does not occur on the crystalline enzyme. When meropenem or ertapenem was reacted with SHV-1 in solution, the Raman difference spectra demonstrated that only a major population corresponding to the Delta (1) acyl-enzyme could be detected. The 1003 cm (-1) mode of the phenyl ring positioned on the C3 side chain of ertapenem acts as an effective internal Raman intensity standard, and the ratio of its intensity to that of the 1600 cm (-1) feature of Delta (1) provides an estimate of the relative populations of Delta (1). In solution, I 1600/ I 1003 equals 2, and in the crystal, I 1600 /I 1003 equals 1. This is strong evidence that the Delta (1) and Delta (2) acyl-enzymes in the crystal are present in approximately equal amounts, in agreement with the X-ray data. However, in solution there are twice as many Delta (1) species per Phe group, and this represents approximately 100% of the active sites, which is consistent with the observed inhibition of the enzyme's activity.
| S-EPMC2656688 | biostudies-literature
Project description:The constitutively expressed, chromosomally encoded β-lactamase (BlaC) is the enzyme responsible for the intrinsic resistance to β-lactam antibiotics in Mycobacterium tuberculosis. Previous studies from this laboratory have shown that the enzyme exhibits an extended-spectrum phenotype, with very high levels of penicillinase and cephalosporinase activity, as well as weak carbapenemase activity [Tremblay, L. W., et al. (2008) Biochemistry 47, 5312-5316]. In this report, we have determined the pH dependence of the kinetic parameters, revealing that the maximal velocity depends on the ionization state of two groups: a general base exhibiting a pK value of 4.5 and a general acid exhibiting a pK value of 7.8. Having defined a region where the kinetic parameters are pH-independent (pH 6.5), we determined solvent kinetic isotope effects (SKIEs) for three substrates whose kcat values differ by 5.5 orders of magnitude. Nitrocefin is a highly activated, chromogenic cephalosporin derivative that exhibits steady-state solvent kinetic isotope effects of 1.4 on both V and V/K. Cefoxitin is a slower cephalosporin derivative that exhibits a large SKIE on V of 3.9 but a small SKIE of 1.8 on V/K in steady-state experiments. Pre-steady-state, stopped-flow experiments with cefoxitin revealed a burst of β-lactam ring opening with associated SKIE values of 1.6 on the acylation step and 3.4 on the deacylation step. Meropenem is an extremely slow substrate for BlaC and exhibits burst kinetics in the steady-state experiments. SKIE determinations with meropenem revealed large SKIEs on both the acylation and deacylation steps of 3.8 and 4.0, respectively. Proton inventories in all cases were linear, indicating the participation of a single solvent-derived proton in the chemical step responsible for the SKIE. The rate-limiting steps for β-lactam hydrolysis of these substrates are analyzed, and the chemical steps responsible for the observed SKIE are discussed.
| S-EPMC3750105 | biostudies-literature
Project description:Optimization by energy minimization of stable complexes occurring along the pathway of hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase has highlighted a proton shuttle that may explain the catalytic mechanism of the beta-lactamases of class A. Five residues, S70, S130, N132, T235 and A237, are involved in ligand binding. The gamma-OH group of T235 and, in the case of benzylpenicillin, the gamma-OH group of S130 interact with the carboxylate group, on one side of the ligand molecule. The side-chain NH2 group of N132 and the carbonyl backbone of A237 interact with the exocyclic CONH amide bond, on the other side of the ligand. The backbone NH groups of S70 and A237 polarize the carbonyl group of the scissile beta-lactam amide bond. Four residues, S70, K73, S130 and E166, and two water molecules, W1 and W2, perform hydrolysis of the bound beta-lactam compound. E166, via W1, abstracts the proton from the gamma-OH group of S70. While losing its proton, the O-gamma atom of S70 attacks the carbonyl carbon atom of the beta-lactam ring and, concomitantly, the proton is delivered back to the adjacent nitrogen atom via W2, K73 and S130, thus achieving formation of the acyl-enzyme. Subsequently, E166 abstracts a proton from W1. While losing its proton, W1 attacks the carbonyl carbon atom of the S70 ester-linked acyl-enzyme and, concomitantly, re-entry of a water molecule W'1 replacing W1 allows E166 to deliver the proton back to the same carbonyl carbon atom, thus achieving hydrolysis of the beta-lactam compound and enzyme recovery. The model well explains the differences found in the kcat. values for hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase. It also explains the effects caused by site-directed mutagenesis of the Bacillus cereus beta-lactamase I [Gibson, Christensen & Waley (1990) Biochem J. 272, 613-619].
| S-EPMC1151568 | biostudies-other
Project description:The structural diversity in metallo-β-lactamases (MBLs), especially in the vicinity of the active site, has been a major hurdle in the development of clinically effective inhibitors. Representatives from three variants of the B3 MBL subclass, containing either the canonical HHH/DHH active-site motif (present in the majority of MBLs in this subclass) or the QHH/DHH (B3-Q) or HRH/DQK (B3-RQK) variations, were reported previously. Here, we describe the structure and kinetic properties of the first example (SIE-1) of a fourth variant containing the EHH/DHH active-site motif (B3-E). SIE-1 was identified in the hexachlorocyclohexane-degrading bacterium Sphingobium indicum, and kinetic analyses demonstrate that although it is active against a wide range of antibiotics, its efficiency is lower than that of other B3 MBLs but has increased efficiency toward cephalosporins relative to other β-lactam substrates. The overall fold of SIE-1 is characteristic of the MBLs; the notable variation is observed in the Zn1 site due to the replacement of the canonical His116 by a glutamate. The unusual preference of SIE-1 for cephalosporins and its occurrence in a widespread environmental organism suggest the scope for increased MBL-mediated β-lactam resistance. Thus, it is relevant to include SIE-1 in MBL inhibitor design studies to widen the therapeutic scope of much needed antiresistance drugs.
| S-EPMC8448093 | biostudies-literature
Project description:Serine-based β-lactamases of Class A, C and D all rely on a key water molecule to hydrolyze and inactivate β-lactam antibiotics. This process involves two conserved catalytic steps. In the first acylation step, the β-lactam antibiotic forms an acyl-enzyme intermediate (ES*) with the catalytic serine residue. In the second deacylation step, an activated water molecule serves as nucleophile (WAT_Nu) to attack ES* and release the inactivated β-lactam. The coordination and activation of WAT_Nu is not fully understood. Using time-resolved x-ray crystallography and QM/MM simulations, we analyzed three intermediate structures of Class A β-lactamase PenP as it slowly hydrolyzed cephaloridine. WAT_Nu is centrally located in the apo structure but becomes slightly displaced away by ES* in the post-acylation structure. In the deacylation structure, WAT_Nu moves back and is positioned along the Bürgi-Dunitz trajectory with favorable energetic profile to attack ES*. Unexpectedly, WAT_Nu is also found to adopt a catalytically incompetent conformation in the deacylation structure forming a hydrogen bond with ES*. Our results reveal that ES* plays a significant role in coordinating and activating WAT_Nu through subtle yet distinct interactions at different stages of the catalytic process. These interactions may serve as potential targets to circumvent β-lactamase-mediated antibiotic resistance.
| S-EPMC7311446 | biostudies-literature
Project description:The pH- and temperature-dependence of steady-state kinetic parameters for 6-beta-(2-furyl)-acryloylamido-penicillanic acid showed it to be a good substrate of staphylococcal PC1 beta-lactamase, and the viscosity-dependence of K(m)/k(cat) indicated that steps up to the formation of the acyl-enzyme were partially diffusion-limited. In the pH range 4-9, a pre-steady-state transient blue shift in the UV absorption spectrum of the bound furyl-acryloylamido chromophore was of constant amplitude and decayed to the spectrum of the product with a first-order rate constant equal to k(cat). The spectrum of the isolated denatured acyl-enzyme was similar to that of the methyl ester of furyl-acryloylpenicilloic acid, pointing to non-covalent interactions with the folded protein, possibly associated with the charge on Glu-166, as the source of the blue-shifted spectrum. Taken together, these results point to a rapid acylation and slower deacylation at Ser-70 and imply that ionization of groups affecting enzyme activity at alkaline pH, for which likely candidates are Lys-73 and Lys-234, affect the rate of deacylation.
| S-EPMC1217229 | biostudies-other
Project description:UnlabelledAngiotensin-1-converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (Aβ) substrates together with high resolution crystal structures of the N-domain in complex with Aβ fragments. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14-Gln15. Furthermore, Aβ(1-16) was preferentially cleaved by the individual N-domain; however, the presence of an inactive C-domain in full-length somatic ACE (sACE) greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7-Ser8 bond with all ACE constructs showing greater catalytic efficiency for Aβ(4-10)Y. Surprisingly, in contrast to Aβ(1-16) and Aβ(4-10)Q, sACE showed positive domain cooperativity and the double C-domain (CC-sACE) construct no cooperativity towards Aβ(4-10)Y. The structures of the Aβ peptide-ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C-terminal P2' position to the S2' pocket and recognizes the main chain of the P1' peptide. It is likely that N-domain selectivity for the amyloid peptide is conferred through the N-domain specific S2' residue Thr358. Additionally, the N-domain can accommodate larger substrates through movement of the N-terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain selectivity are more pronounced with the truncated domains compared to the more physiological full-length forms.DatabaseThe atomic coordinates and structure factors for N-domain ACE with Aβ peptides 4-10 (5AM8), 10-16 (5AM9), 1-16 (5AMA), 35-42 (5AMB) and (4-10)Y (5AMC) complexes have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/).
| S-EPMC4950319 | biostudies-literature