Biomedical subjects

R F Pratt

Publications and source records attributed to R F Pratt.

At least 37 records · Page 2Linked to original sources

PubMed2003

Hydrolysis of third-generation cephalosporins by class C beta-lactamases. Structures of a transition state analog of cefotoxamine in wild-type and extended spectrum enzymes.

Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics. An important source of this resistance is from production of extended-spectrum (ES) beta-lactamases by bacteria. The Enterobacter cloacae GC1 enzyme is an example of a class C ES beta-lactamase. Unlike wild-type (WT) forms, such as the E. cloacae P99 and Citrobacter freundii enzymes, the ES GC1 beta-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime. To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-[(Z)-methoxyimino]acetylaminomethyl phosphonate has been synthesized and characterized. This phosphonate was designed to generate a transition state analog for turnover of cefotaxime. The crystal structures of complexes of the phosphonate with both ES GC1 and WT C. freundii GN346 beta-lactamases have been determined to high resolution (1.4-1.5 Angstroms). The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme. In the WT beta-lactamase the cefotaxime-like side chain is crowded against the Omega loop and must protrude from the binding site with its methyloxime branch exposed. In the ES enzyme, a mutated Omega loop adopts an alternate conformation allowing the side chain to be much more buried. During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule. The ES beta-lactamase is thus able to efficiently inactivate third-generation cephalosporins.

Amino Acid Sequence↗

PubMed2003

New substrates for beta-lactam-recognizing enzymes: aryl malonamates.

Aryl malonamates are demonstrated to be novel substrates of a broad range of beta-lactam-recognizing enzymes. These compounds are isomers of the aryl phenaceturates, which are well-known substrates of these enzymes, but the new compounds contain a retro-amide side chain. Several lines of evidence, including comparisons of steady-state kinetic parameters between enzymes and a detailed investigation of the methanolysis kinetics, solvent deuterium isotope effects, and pH-rate profile for turnover of a retro substrate by the Enterobacter cloacae P99 beta-lactamase, suggested that the new substrates are likely to be hydrolyzed by the same chemical mechanisms as "normal" substrates. Molecular modeling indicated that the retro-amide group fits snugly into the active site of the P99 beta-lactamase by hydrogen bonding to the conserved lysine-67 residue. The retro-amide side chain may represent a lead to novel mechanism-based and transition state analogue inhibitors.

Acylation↗

PubMed2003

Inhibition of beta-lactamases by monocyclic acyl phosph(on)ates.

The cyclic acyl phosph(on)ates, 1-hydroxy-5-phenyl-2,6-dioxaphosphorinone(3)-1-oxide, its 4-phenyl isomer, and the phosphonate (2-oxo) analogue of the latter inhibited typical class A (TEM-2) and class C (Enterobacter cloacae P99) beta-lactamases in a time-dependent fashion. No enzyme-catalyzed turnover was detected in any case. The interactions occurring were interpreted in terms of the reaction scheme E + I left arrow over right arrow EI left arrow over right arrow EI', where EI is a reversibly formed noncovalent complex, and EI' is a covalent complex. Reactions of the cyclic phosphates with the P99 beta-lactamase were effectively irreversible, while that of the 4-phenyl cyclic phosphate with the TEM beta-lactamase was slowly reversible. The 4-phenyl cyclic phosphate was generally the most effective inhibitor, both kinetically and thermodynamically, with second-order rate constants of inactivation of both enzymes around 10(4) s(-1) M(-1). This compound also bound noncovalently to both enzymes, with dissociation constants of 25 microM from the P99 enzyme and 100 microM from the TEM. It is unusual to find an inhibitor equally effective against the TEM and P99 enzymes; the beta-lactamase inhibitors currently employed in medical practice (e.g., clavulanic acid) are significantly more effective against class A enzymes. The results of lysinoalanine analysis after hydroxide treatment of the inhibited enzymes and of a (31)P nuclear magnetic resonance spectrum of one such complex were interpreted as favoring a mechanism of inactivation by enzyme acylation rather than phosphylation. Molecular modeling of the enzyme complexes of the 4-phenyl phosphate revealed bound conformations where recyclization and thus reactivation of the enzyme would be difficult. The compounds studied were turned over slowly or not at all by acetylcholinesterase and phosphodiesterase I.

Acetylcholinesterase↗

PubMed2003

The crystal structure of phosphonate-inhibited D-Ala-D-Ala peptidase reveals an analogue of a tetrahedral transition state.

D-Alanyl-D-alanine carboxypeptidase/transpeptidases (DD-peptidases) are beta-lactam-sensitive enzymes that are responsible for the final peptidoglycan cross-linking step in bacterial cell wall biosynthesis. A highly specific tripeptide phosphonate inhibitor was designed with a side chain corresponding to a portion of the Streptomyces R61 peptidoglycan. This compound was found to be a slow, irreversible inactivator of the DD-peptidase. Molecular modeling suggested that although a pentacoordinated intermediate of the phosphonylation reaction would not interact strongly with the enzyme, a tetracoordinated phosphonyl enzyme might be analogous to a transition state in the reaction with peptide substrates. To investigate this possibility, the crystal structure of the phosphonyl enzyme was determined. The 1.1 A resolution structure shows that the inhibitor has phosphonylated the catalytic serine (Ser62). One of the phosphonyl oxygens is noncovalently bound in the oxyanion hole, while the other is solvated by two water molecules. The conserved hydroxyl group of Tyr159 forms a strong hydrogen bond with the latter oxygen atom (2.77 A). This arrangement is interpreted as being analogous to the transition state for the formation of the tetrahedral intermediate in the deacylation step of the carboxypeptidase reaction. The proximity of Tyr159 to the solvated phosphonyl oxygen suggests that the tyrosine anion acts as a general base for deacylation. This transition state analogue structure is compared to the structures of noncovalent DD-peptidase reaction intermediates and phosphonylated beta-lactamases. These comparisons show that specific substrate binding to the peptidase induces a conformational change in the active site that places Ser62 in an optimal position for catalysis. This activated conformation relaxes as the reaction proceeds.

Binding Sites↗

PubMed2002

Structures of two kinetic intermediates reveal species specificity of penicillin-binding proteins.

Penicillin-binding proteins (PBPs), the target enzymes of beta-lactam antibiotics such as penicillins and cephalosporins, catalyze the final peptidoglycan cross-linking step of bacterial cell-wall biosynthesis. beta-Lactams inhibit this reaction because they mimic the D-alanyl-D-alanine peptide precursors of cell-wall structure. Prior crystallographic studies have described the site of beta-lactam binding and inhibition, but they have failed to show the binding of D-Ala-D-Ala substrates. We present here the first high-resolution crystallographic structures of a PBP, D-Ala-D-Ala-peptidase of Streptomyces sp. strain R61, non-covalently complexed with a highly specific fragment (glycyl-L-alpha-amino-epsilon-pimelyl-D-Ala-D-Ala) of the cell-wall precursor in both enzyme-substrate and enzyme-product forms. The 1.9A resolution structure of the enzyme-substrate Henri-Michaelis complex was achieved by using inactivated enzyme, which was formed by cross-linking two catalytically important residues Tyr159 and Lys65. The second structure at 1.25A resolution of the uncross-linked, active form of the DD-peptidase shows the non-covalent binding of the two products of the carboxypeptidase reaction. The well-defined substrate-binding site in the two crystallographic structures shows a subsite that is complementary to a portion of the natural cell-wall substrate that varies among bacterial species. In addition, the structures show the displacement of 11 water molecules from the active site, the location of residues responsible for substrate binding, and clearly demonstrate the necessity of Lys65 and or Tyr159 for the acylation step with the donor peptide. Comparison of the complexed structures described here with the structures of other known PBPs suggests the design of species-targeted antibiotics as a counter-strategy towards beta-lactamase-elicited bacterial resistance.

Bacterial Proteins↗

PubMed2002

Formation and structure of 1:1 complexes between aryl hydroxamic acids and vanadate at neutral pH.

Although aryl hydroxamic acids are well-known to form coordination complexes with vanadate (V(V)), the nature of these complexes at neutral pH and submillimolar concentrations, the conditions under which such complexes inhibit various serine amidohydrolases, is not well established. A series of qualitative and quantitative experiments, involving UV/vis, (1)H NMR, and (51)V NMR spectroscopies, established that both 1:1 and 1:2 vanadate/hydroxamate complexes form at pH 7.5, with the former dominating at submillimolar concentrations. Formation constants for the complexes of several aryl and alkyl hydroxamic acids were determined; for example, for benzohydroxamic acid, the stepwise formation constants of the 1:1 and 1:2 complexes were 3000 and 400 M(-1), respectively. The (51)V chemical shift of the 1:1 4-nitrobenzohydroxamic acid complex was -497 ppm, and that of its unsubstituted analogue was -498 ppm. A (1)H-(15)N HSQC spectrum of the 4-nitrobenzo-(15)N-hydroxamic acid/vanadate complex indicated the presence of an N-H group with (15)N and (1)H chemical shifts of 115 and 5.83 ppm, respectively. A (13)C NMR spectrum of the complex of 4-nitrobenzo-(13)C-hydroxamic acid with vanadate displayed a resonance at 170.1 ppm and thus a coordination-induced shift (CIS) of +3.8 ppm. In contrast, the CIS value of an established 1:2 complex, thought to contain chelated hydroxamic acid ligands, was +11.9 ppm. These spectral data led to the following structural picture of 1:1 complexes of vanadate and aryl hydroxamic acids. They contain penta- or hexa-coordinated vanadium. The ligand is in the hydroxamate rather than hydroximate form. The ligand is presumably bound to vanadium through the hydroxamic hydroxyl oxygen, but the hydroxamic acid carbonyl oxygen interacts weakly with vanadium. These species are the most likely candidates for the inhibitors of serine amidohydrolases found in vanadate/hydroxamic acid mixtures.

Hydrogen-Ion Concentration↗

PubMed2002

Mechanism of inhibition of the beta-lactamase of Enterobacter cloacae P99 by 1:1 complexes of vanadate with hydroxamic acids.

The class C beta-lactamase of Enterobacter cloacae P99 is competitively inhibited by low concentrations of 1:1 complexes of vanadate and hydroxamic acids. Structure-activity studies indicated that the hydroxamic acid functional group was essential to this inhibition. Both aryl and alkyl hydroxamic acids form inhibitory ternary complexes with vanadate and the enzyme, although, in certain cases of the latter, the inhibition may not be seen because of the low formation constants of the vanadate-hydroxamic acid complex. After all of the vanadate species present in solution had been taken into account, "real" K(i) values for the vanadate complexes could be determined. The K(i) value of the best of the inhibitors that were investigated, the 1:1 complex of vanadate with 4-nitrobenzohydroxamic acid, was 0.48 microM. Kinetics studies showed that the association and dissociation rate constants of this complex with the enzyme were 1.48 x 10(6) s(-1) M(-1) and 0.73 s(-1), respectively; the magnitude of the latter indicates covalent interaction of the complex with the enzyme. (51)V NMR and UV-vis spectra suggest that the structure of the vanadate complex bound to the enzyme may be very similar to that in solution. A (13)C NMR spectrum of the enzyme complex with 4-nitrobenzo[(13)C]hydroxamic acid and vanadate yields a coordination-induced shift (CIS) of 7.74 ppm. This is significantly larger than that of the vanadate complex in free solution (3.62 ppm), suggesting either, somewhat contrary to the (51)V and UV-vis spectra, greater interaction between vanadium and the hydroxamate carbonyl oxygen in the enzyme complex than in free solution or, more likely, polarization of the hydroxamate by interaction, e.g., hydrogen bonding, with the enzyme. Molecular modeling indicates that a pentacoordinated vanadate complex may well be able to snugly occupy the enzyme active site; Asn 152 is suitably placed to hydrogen bond to the hydroxamic acid oxygen atom. The experimental results are in accord with a model whereby the vanadate-hydroxamate-enzyme complex is a moderately good analogue of the transition state of the reaction of the beta-lactamase with phosphonate inhibitors.

Dose-Response Relationship, Drug↗

PubMed2001

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by cyclic acyl phosph(on)ates: rescue by return.

As previously described (Pratt, R. F.; Hammar, N. J. J. Am. Chem. Soc. 1998, 120, 3004.), 1-hydroxy-4,5-benzo-2,6-dioxaphosphorinone(3)-1-oxide (salicyloyl cyclic phosphate) inactivates the class C beta-lactamase of Enterobacter cloacae P99 in a covalent fashion. The inactivated enzyme slowly reverts to the active form. This paper shows that reactivation involves a recyclization reaction that regenerates salicyloyl cyclic phosphate rather than hydrolysis of the covalent intermediate. The inactivation, therefore, is a slowly reversible covalent modification of the active site. The thermodynamic dissociation constant of the inhibitor from the inactivated enzyme is 0.16 microM. Treatment of the inactivated enzyme with alkali does not produce salicylic acid but does, after subsequent acid hydrolysis, yield one molar equivalent of lysinoalanine. This result proves that salicyloyl cyclic phosphate inactivates the enzyme by (slowly reversible) phosphorylation of the active site serine residue. This result contrasts sharply with the behavior of acyclic acyl phosphates which transiently inactivate the P99 beta-lactamase by acylation (Li, N.; Pratt, R. F. J. Am. Chem. Soc. 1998, 120, 4264.). This chemoselectivity difference is explored by means of molecular modeling. Rather counterintuitively, in view of the relative susceptibility of phosphates and phosphonates to nucleophilic attack at phosphorus, 1-hydroxy-4,5-benzo-2-oxaphosphorinanone(3)-1-oxide, the phosphonate analogue of salicyloyl cyclic phosphate, did not appear to inactivate the P99 beta-lactamase in a time-dependent fashion. It was found, however, to act as a fast reversible inhibitor (K(i) = 10 microM). A closer examination of the kinetics of inhibition revealed that both on and off rates (9.8 x 10(3) s(-1) x M(-1) and 0.098 s(-1), respectively) were much slower than expected for noncovalent binding. This result strongly indicates that the inhibition reaction of the phosphonate also involves phosphylation of the active site. Hence, unlike the situation with bacterial DD-peptidases covalently inactivated by beta-lactams, the P99 beta-lactamase inactivated by the above cyclic acyl phosph(on)ates can be rescued by return. Elimination of the recyclization reaction would lead to more effective inhibitors.

Enterobacter cloacae↗

PubMed2001

Mechanism of reaction of acyl phosph(on)ates with the beta-lactamase of Enterobacter cloacae P99.

A series of acyl phosph(on)ates has been prepared to more closely examine the details of the interactions of this class of molecule with beta-lactamases. In general, they were found to react with the class C beta-lactamase of Enterobacter cloacae P99 in two ways, by acylation and by phosphylation. The acyl-enzymes generated by the former reaction were transiently stable with half-lives of between 3 and 45 s, of comparable lifetime therefore to those generated by the inhibitory beta-lactams cefotaxime, cefuroxime, and cefoxitin. On the other hand, phosphylation led to a completely inactive enzyme. In general, the second-order rate constants for acylation (k(cat)/K(m)) were larger than for phosphylation (k(i)). As expected on chemical grounds, phosphylation was found to be relatively more effective for the phosphonates than the phosphates. The acyl phosphates were much more effective acylating agents however. The acylation reaction was found to be enhanced by hydrophobic substituents in both the acyl and leaving group moieties. Thus, the most reactive compound in this series was benzo[b]thiophene-2-carbonyl 2'-naphthyl phosphate with a K(m) value of 0.15 microM and a k(cat) of 0.2 s(-1); k(cat)/K(m) is therefore 1.3 x 10(6) s(-1) M(-1), making this compound the most specific acyclic acylation reagent for this beta-lactamase yet described. Significant substrate inhibition by this compound suggested that further binding regions may be available for exploitation in inhibitor design. A linear free energy analysis showed that the transition states for acylation of the beta-lactamase by aroyl phosphates are analogues of the corresponding aryl boronic acid adducts. Molecular modeling suggested that the aroyl phosphates react with the P99 beta-lactamase with the aroyl group in the side chain/acyl group site of normal substrates and the phosphate in the leaving group site. In this orientation, the phosphate leaving group interacts strongly with Lys 315.

Acylation↗

PubMed2001

Inverse acyl phosph(on)ates: substrates or inhibitors of beta-lactam-recognizing enzymes?

Acyl phosph(on)ates represent a new class of inhibitors of beta-lactam-recognizing enzymes. Previously described members of this class were aroyl phosph(on)ates. These compounds have been shown to acylate and/or phosphylate the active site serine residue, leading to either transient or essentially irreversible inhibition [Li, N., and Pratt, R. F. (1998) J. Am. Chem. Soc.120, 4264-4268]. The present paper describes the synthesis and evaluation as inhibitors of an inverse pair of acyl phosph(on)ates that incorporate the amido side chain that represents a major substrate specificity determinant of these enzymes. Thus, N-(phenylacetyl)glycyl phenyl phosphate and benzoyl N-(benzyloxycarbonyl)aminomethyl phosphonate were prepared. The former of these compounds was found to be a substrate of typical class A and C beta-lactamases and of the DD-peptidase of Streptomyces R61; it thus acylates the active site serine. In contrast, the latter compound was an irreversible inhibitor of the above enzymes, probably by phosphonylation of the active site serine. With each of these enzymes therefore, the amido side chain rather than the acyl group dictates the orientation of the bound phosph(on)ate and thus the mode of reaction.

Benzoates↗

PubMed2001

The synthesis and evaluation of benzofuranones as beta-lactamase substrates.

6- and 7-Carboxy-3-phenylacetamido-3H-1-benzofuran-2-one have been synthesized as potential beta-lactamase substrates and/or inhibitors. These compounds were prepared by lactonization of the corresponding, appropriately substituted phenylglycines. The latter compounds were prepared by either the Strecker or the Bücherer-Berg method. The benzofuran-2-ones were less stable in aqueous solution than the analogous acyclic phenaceturate esters but comparably stable to analogous benzopyran-2-ones. They differed from the latter compounds however in that the C-3 hydrogen of the furan-2-ones, adjacent to the lactone carbonyl group, was distinctly acidic; 7-carboxy-3-phenylacetamido-3H-1-benzofuran-2-one exists largely as an enolate at pH 7.5. The furan-2-ones were beta-lactamase substrates with reactivity very similar to the analogous acyclic phenaceturates. They were not, however, DD-peptidase inhibitors and are thus unlikely to have antibiotic activity. The structural basis for these observations is discussed.

Benzofurans↗

PubMed2001

Design, synthesis, and evaluation of alpha-ketoheterocycles as class C beta-lactamase inhibitors.

A series of specific alpha-ketoheterocycles (benzoxazole, thiazole, imidazole, tetrazole, and thiazole-4-carboxylate) has been synthesized in order to assess their potential as beta-lactamase inhibitors. The syntheses were achieved either by construction of the heterocycle (benzoxazole) from an appropriate alpha-hydroxyimidate, followed by oxidation of the alcohol, or by direct reaction of methyl phenaceturate with a lithiated heterocycle. The properties of these compounds in aqueous solution are described and their inhibitory activity against beta-lactamases assessed. They did inhibit the class C beta-lactamase of Enterobacter cloacae P99 but not the TEM beta-lactamase. The most effective inhibitor of the former enzyme (K(i)=0.11 mM) was 5-(phenylacetylglycyl) tetrazole, probably because it is an anion at neutral pH. Interpretation of the results was aided by computational models of the tetrahedral adducts. Most of the compounds also inhibited alpha-chymotrypsin but not porcine pancreatic elastase.

Drug Design↗

PubMed2001

CENTA as a chromogenic substrate for studying beta-lactamases.

CENTA, a chromogenic cephalosporin, is readily hydrolyzed by beta-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of beta-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.

Cephalosporins↗

PubMed2000

Dipeptide binding to the extended active site of the Streptomyces R61 D-alanyl-D-alanine-peptidase: the path to a specific substrate.

Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-D-alanine(DD)-peptidases/transpeptidases. The natural substrates of these enzymes should therefore be segments of peptidoglycan, but high specificity for such structures has yet to be demonstrated. The binding of dipeptides to the extended substrate binding site of the Streptomyces R61 DD-peptidase has been studied by means of a fluorescent beta-lactam probe. It was found that dipeptides of structure Gly-L-Xaa have affinity for a subsite adjacent to the beta-lactam binding site. Hydrophobic peptides such as Gly-L-Met and Gly-L-aminocaprylic acid had the greatest affinity for this site, with dissociation constants in each case of 0.19 mM. A combination of this motif with the C-terminal D-alanyl-D-alanine moiety required of a DD-peptidase substrate yielded a new substrate, glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine. Steady-state kinetic measurements established this compound as the most specific peptide substrate yet discovered for a DD-peptidase by at least 3 orders of magnitude (k(cat) = 69 s(-1), K(m) = 7.9 microM, k(cat)/K(m) = 8.7 x 10(6) s(-1) M(-1)); acylation was rate-determining at saturation. This substrate, presumably not coincidentally, contains the acyl donor and acceptor moieties, appropriately separated, of the Streptomyces peptidoglycan structure. This general method of approach should be of value in the search for specific substrates and inhibitors (antibiotics) of other DD-peptidases.

Binding Sites↗

PubMed2000

Inhibition of serine amidohydrolases by complexes of vanadate with hydroxamic acids.

Serine beta-lactamases are inhibited by phosphonate monoester monoanions. These compounds phosphonylate the active site serine hydroxyl group to form inert, covalent complexes. Since spontaneous hydrolysis of these phosphonates is generally quite slow, the beta-lactamase active site must have considerable affinity for the (presumably) pentacoordinated phosphonyl transfer transition state. Structural analogs of such a transition state might well therefore be effective and novel beta-lactamase inhibitors. Complexes of vanadate with hydroxamic acids may be able to achieve such a structure. Indeed, mixtures of these two components, but neither one alone, were found to inhibit a typical class C beta-lactamase. A Job plot of the inhibition by vanadate/benzohydroxamic acid mixtures indicated that the inhibitor was a 1:1 complex for which an inhibition constant of 4.2 microM could be calculated. A bacterial DD-peptidase, structurally similar to the beta-lactamase, was also inhibited (K(i) = 22 microM) by this complex. A similar rationale would suggest that other serine hydrolases might also be inhibited by these mixtures. In fact, chymotrypsin was inhibited by a complex of vanadate with benzohydroxamic acid (K(i) = 10 microM) and elastase by a complex with acetohydroxamic acid (K(i) = 90 microM).

Bacterial Proteins↗

PubMed2000

The Oxyanion Hole in Serine beta-Lactamase Catalysis: Interactions of Thiono Substrates with the Active Site.

Both functional and structural studies of serine beta-lactamases indicate the existence of an oxyanion hole at the active site with an important role in catalysis. The functional presence of the oxyanion hole is demonstrated by the previous observation that thiono-beta-lactams are very poor substrates of beta-lactamases (B. P. Murphy, and R. F. Pratt, 1988, Biochem. J. 256, 669-672) and in the present paper by the inability of these enzymes to catalyze hydrolysis of a thiono analog of a depsipeptide substrate. This thiono effect was first noted and interpreted in regard to classical serine hydrolases although the chemical basis for it has not been firmly established either in those enzymes or in beta-lactamases. In this paper a computational approach to a further understanding of the effect has been taken. The results for a class C beta-lactamase show that the deacylation tetrahedral intermediate interacted more strongly with the enzyme with an O(-) placed in the oxyanion hole than an S(-). On the other hand, the converse was true for acylation tetrahedral intermediate species, a result distinctly not in accord with experiment. These results indicate that the thiono effect does not arise from unfavorable interactions between enzyme and thiono substrate at the tetrahedral intermediate stage but must be purely kinetic in nature, i.e., arise in a transitional species at an early stage of the acylation reaction. The same conclusion as to the origin of the thiono effect was also indicated by a less extensive series of calculations on a class A beta-lactamase and on chymotrypsin.

Journal Article↗

PubMed1999

Interactions of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with moenomycin.

Kinetics studies in homogeneous aqueous solution showed that solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus (a bacterial DD-peptidase) was inhibited by the amphiphilic glycolipid antibiotic moenomycin. Inhibition at the peptidase site was determined by competition experiments between moenomycin and the chromophoric beta-lactam nitrocefin. Under conditions of high salt concentration (1 M NaCl), pseudo-first-order rate constants for the reaction of moenomycin with sPBP2a leading to inhibition of acylation by nitrocefin varied with moenomycin concentration in a biphasic fashion. At low moenomycin concentration (<20 microM) little inhibition occurred, but at higher concentrations a linear increase in rate constant with moenomycin concentration was observed, yielding a second-order rate constant of inhibition of 120 s(-)(1) M(-)(1). Since the cmc of moenomycin under these conditions was shown to be ca. 20 microM, the inhibition was concluded to arise from reaction of sPBP2a with a moenomycin micelle. Protein fluorescence studies showed a pseudo-first-order decrease in fluorescence on reaction of the protein with moenomycin. The variation of this rate constant with moenomycin concentration was consistent with reaction of a moenomycin monomer with the protein with a second-order rate constant of 650 s(-)(1) M(-)(1). This monomer reaction did not occur at the DD-peptidase site since its rate was unaffected by prior acylation of the enzyme by benzylpenicillin; nor did it inhibit reaction at that site by beta-lactams. Under low salt conditions (0.175 M NaCl) where reaction could be studied over a greater range of monomer concentrations since the cmc was ca. 120 microM, similar reactions were involved. Under these circumstances, inhibition was concerted with the reaction of moenomycin monomers, although fast premicellar aggregation of moenomycin with the protein also occurred. All moenomycin interactions with sPBP2a were reversible, as revealed by detergent-extraction chromatography. Lower limits to moenomycin off-rates and equilibrium dissociation constants were 7.7 x 10(-)(4) s(-)(1) and 1.2 microM, respectively. Other amphiphiles did not react in exactly the same manner as moenomycin, indicating some degree of specificity in reactions of the latter. sPBP2a did not have detectable affinity for lipid surfaces (Triton X-114 and phosphatidylglycerol vesicles). A general scheme for reaction of moenomycin with sPBP2a is proposed.

Acylation↗

PubMed1999

On the importance of a methyl group in beta-lactamase evolution: free energy profiles and molecular modeling.

beta-Lactam antibiotics are generally thought to inhibit their target enzymes, the bacterial cell wall-synthesizing DD-peptidases, because of their resemblance to D-alanyl-D-alanine peptides. Although a favorable conformation of the latter does structurally resemble the beta-lactams with respect to backbone conformation, a significant difference is the presence of a D-methyl substituent on the penultimate alanine residue of the cell wall peptide. A classical beta-lactam antibiotic has a hydrogen in the corresponding position. In the process of evolution of a beta-lactamase from a DD-peptidase, it seems likely that this D-methyl group would be selected against, to ensure that the former enzyme would hydrolyze beta-lactams rather than peptides. In this paper, the effect of the penultimate D-alanine residue (as opposed to a glycine residue) has been examined in peptide substrates of a present-day DD-peptidase and a beta-lactamase. The peptides N-(phenylacetyl)-D-alanyl-D-phenylalanine and N-(phenylacetyl)glycyl-D-phenylalanine were used as a test pair against the DD-peptidase of Streptomyces R61 and the structurally very similar class C beta-lactamase of Enterobacter cloacae P99. The kinetics of turnover of both of these substrates were determined for both enzymes. To quantify the partitioning of the acyl-enzyme intermediate, the aminolysis by D-phenylalanine of a cognate pair of depsipeptides was also studied. Thus, free energy-reaction coordinate diagrams were constructed for turnover of both peptides by both enzymes. Comparison of these profiles showed that the D-methyl group is preferred over hydrogen by the DD-peptidase at all stages of catalysis (acyl-enzyme and acylation and deacylation transition states), whereas the beta-lactamase selects against the D-methyl group only at the peptide acylation transition state. A process of evolution by uniform dissociation of the methyl group by the beta-lactamase has apparently occurred. These results were explored structurally by computational models of the acylation tetrahedral intermediates. A methyl group pocket on the DD-peptidase, less favorable on the beta-lactamase, was identified. The interaction of the leaving group, the terminal D-alanine residue, with the two enzymes was interesting, since it seemed that different positively charged active site residues were directly associated with the carboxylate, Lys 315 in the beta-lactamase and Arg 285 (rather than His 298) in the case of the DD-peptidase. The problems posed by larger substituents on the penultimate residue of the peptide, and in particular by the heterocyclic substituent present in a bicyclic beta-lactam, were analyzed. Qualitative and quantitative analysis of the models support the proposed importance of the penultimate D-alanine in beta-lactamase evolution.

Carboxypeptidases↗