Detection of an enzyme isomechanism by means of the kinetics of covalent inhibition.

Turnover of substrates by many enzymes involves free enzyme forms that differ from the stable form of the enzyme in the absence of substrate. These enzyme species, known as isoforms, have, in general, different physical and chemical properties than the native enzymes. They usually occur only in small concentrations under steady state turnover conditions and thus are difficult to detect. We show in this paper that in one particular case of an enzyme (a class C ß-lactamase) with specific substrates (cephalosporins) the presence of an enzyme isoform (E') can be detected by means of its different reactivity than the native enzyme (E) with a class of covalent inhibitors (phosphonate monoesters). Generation of E' from E arises either directly from substrate turnover or by way of a branched path from an acyl-enzyme intermediate. The relatively slow spontaneous restoration of E from E' is accelerated by certain small molecules in solution, for example cyclic amines such as imidazole and salts such as sodium chloride. Solvent deuterium kinetic isotope effects and the effect of methanol on cephalosporin turnover showed that for both E and E', kcat is limited by deacylation of an acyl-enzyme intermediate rather than by enzyme isomerization.

Elusive structural changes of New Delhi metallo-ß-lactamase revealed by ultraviolet photodissociation mass spectrometry.

We use mass spectrometry (MS), under denaturing and non-denaturing solution conditions, along with ultraviolet photodissociation (UVPD) to characterize structural variations in New Delhi metallo-ß-lactamase (NDM) upon perturbation by ligands or mutation. Mapping changes in the abundances and distributions of fragment ions enables sensitive detection of structural alterations throughout the protein. Binding of three covalent inhibitors was characterized: a pentafluorphenyl ester, an O-aryloxycarbonyl hydroxamate, and ebselen. The first two inhibitors modify Lys211 and maintain dizinc binding, although the pentafluorophenyl ester is not selective (Lys214 and Lys216 are also modified). Ebselen reacts with the sole Cys (Cys208) and ejects Zn2 from the active site. For each inhibitor, native UVPD-MS enabled simultaneous detection of the closing of a substrate-binding beta-hairpin loop, identification of covalently-modified residue(s), reporting of the metalation state of the enzyme, and in the case of ebselen, observation of the induction of partial disorder in the C-terminus of the protein. Owing to the ability of native UVPD-MS to track structural changes and metalation state with high sensitivity, we further used this method to evaluate the impact of mutations found in NDM clinical variants. Changes introduced by NDM-4 (M154L) and NDM-6 (A233V) are revealed to propagate through separate networks of interactions to direct zinc ligands, and the combination of these two mutations in NDM-15 (M154L, A233V) results in additive as well as additional structural changes. Insight from UVPD-MS helps to elucidate how distant mutations impact zinc affinity in the evolution of this antibiotic resistance determinant. UVPD-MS is a powerful tool capable of simultaneous reporting of ligand binding, conformational changes and metalation state of NDM, revealing structural aspects of ligand recognition and clinical variants that have proven difficult to probe.

A Lysine-Targeted Affinity Label for Serine-ß-Lactamase Also Covalently Modifies New Delhi Metallo-ß-lactamase-1 (NDM-1).

The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-ß-lactamases hamper the development of wide-spectrum ß-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-ß-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-ß-lactamases have an analogous active-site Lys residue used to bind ß-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-ß-lactamases can also serve as a classical affinity label for New Delhi metallo-ß-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 µM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-ß-lactamases.

Specificity of extended O-aryloxycarbonyl hydroxamates as inhibitors of a class C ß-lactamase.

Class C ß-lactamases have previously been shown to be efficiently inactivated by O-aryloxycarbonyl hydroxamates. O-Phenoxycarbonyl-N-benzyloxycarbonylhydroxylamine (1) and O-phenoxycarbonyl-N-(R)-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine (2), for example, were found to be effective inactivators. The present paper describes a structure-activity study of these molecules to better define the important structural elements for high inhibitory activity. The results show that a well-positioned hydrophobic element (which may interact with the Tyr221 residue of the enzyme) and a negatively charged element, e.g. a carboxylate group (which may interact with Arg204), are required for high reactivity with the enzyme. The new compounds were found to inactivate by forming a carbonyl cross-linked enzyme (probably Ser64OCONHLys 315) as for 1 rather than the inert hydroxamoyl derivative observed with 2.

Kinetic Evidence for a Second Ligand Binding Site on Streptococcus pneumoniae Penicillin-Binding Protein 2x.

High molecular mass penicillin-binding proteins (PBPs, DD-peptidases) of class B, such as Streptococcus pneumoniae PBP2x, catalyze the cross-linking of peptidoglycan in bacterial cell wall biosynthesis and are thus important antibiotic targets. Despite their importance in this regard, structure-function studies of ligands of these enzymes have been impeded by the absence of useful substrates. In vitro, these enzymes do not catalyze peptide hydrolysis or aminolysis, their in vivo reaction, but some, such as PBP2x, do catalyze these reactions of certain thioesters such as PhCH2CONHCH2COSCH(D-Me)CO2- (2). We have now prepared several peptidoglycan-mimetic thioesters that we expected to more closely resemble the natural substrates of these enzymes. To our surprise, however, these compounds, although indeed substrates of PBP2x, did not, unlike 2, appear to form an acyl-enzyme intermediate during hydrolysis, and their turnover was inhibited by certain peptides and N-acylamino acids much more weakly than that of 2. An inhibitor of this type, N-benzyloxycarbonyl-d-glutamic acid, also quenched the fluorescence of PBP2x that had been labeled at the DD-peptidase active site by 6-dansylamidopenicillanic acid. These results were interpreted in terms of a model where the peptidoglycan-mimetic thioesters preferentially bound to and hydrolyzed at a site other than the classical DD-peptidase active site. This second site is likely to represent part of an extended binding site that accommodates a peptidoglycan substrate or regulator in vivo. Such a site may be a target for future inhibitor/antibiotic design.

Specificity and mechanism of mandelamide hydrolase catalysis.

The best-studied amidase signature (AS) enzyme is probably fatty acid amide hydrolase (FAAH). Closely related to FAAH is mandelamide hydrolase (MAH), whose substrate specificity and mechanism of catalysis are described in this paper. First, we developed a convenient chromogenic substrate, 4-nitrophenylacetamide, for MAH. The lack of reactivity of MAH with the corresponding ethyl ester confirmed the very limited size of the MAH leaving group site. The reactivity of MAH with 4-nitrophenyl acetate and methyl 4-nitrophenyl carbonate, therefore, suggested formation of an "inverse" acyl-enzyme where the small acyl-group occupies the normal leaving group site. We have interpreted the specificity of MAH for phenylacetamide substrates and small leaving groups in terms of its active site structure, using a homology model based on a FAAH crystal structure. The relevant structural elements were compared with those of FAAH. Phenylmethylboronic acid is a potent inhibitor of MAH (Ki = 27 nM), presumably because it forms a transition state analogue structure with the enzyme. O-Acyl hydroxamates were not irreversible inactivators of MAH but some were found to be transient inhibitors.

Penicillin acylase and O-aryloxycarbonyl hydroxamates: Two acyl-enzymes, one leading to hydrolysis, the other to inactivation.

O-Aryloxycarbonyl hydroxamates have previously been shown to covalently inactivate serine/amine amidohydrolases such as class C ß-lactamases and a N-terminal hydrolase, the proteasome. We report here reactions between O-aryloxycarbonyl hydroxamates and another N-terminal hydrolase, penicillin acylase. O-Aryloxycarbonyl hydroxamates, as non-symmetric carbonates, have two different leaving groups attached to the reactive central carbonyl group. We propose that these compounds can bind to the active site in either of two orientations and that either leaving group can be displaced from either orientation. In the present case we detected from kinetics experiments two distinct acyl-enzymes, one of which is subject to normal hydrolysis and the other to inactivation. Non-symmetric carbonates therefore can be very versatile enzyme inactivators.

Synthesis and Kinetic Analysis of Two Conformationally Restricted Peptide Substrates of Escherichia coli Penicillin-Binding Protein 5.

Escherichia coli PBP5 (penicillin-binding protein 5) is a dd-carboxypeptidase involved in bacterial cell wall maturation. Beyond the C-terminal d-alanyl-d-alanine moiety, PBP5, like the essential high-molecular mass PBPs, has little specificity for other elements of peptidoglycan structure, at least as elicited in vitro by small peptidoglycan fragments. On the basis of the crystal structure of a stem pentapeptide derivative noncovalently bound to E. coli PBP6 (Protein Data Bank entry 3ITB ), closely similar in structure to PBP5, we have modeled a pentapeptide structure at the active site of PBP5. Because the two termini of the pentapeptide are directed into solution in the PBP6 crystal structure, we then modeled a 19-membered cyclic peptide analogue by cross-linking the terminal amines by succinylation. An analogous smaller, 17-membered cyclic peptide, in which the l-lysine of the original was replaced by l-diaminobutyric acid, could also be modeled into the active site. We anticipated that, just as the reactivity of stem peptide fragments of peptidoglycan with PBPs in vivo may be entropically enhanced by immobilization in the polymer, so too would that of our cyclic peptides with respect to their acyclic analogues in vitro. This paper describes the synthesis of the peptides described above that were required to examine this hypothesis and presents an analysis of their structures and reaction kinetics with PBP5.

ß-Lactamases: Why and How.

The targets of ß-lactam antibiotics are bacterial DD-peptidases that catalyze the final steps of peptidoglycan biosynthesis. Bacterial resistance to ß-lactams is achieved by the production of ß-lactamases, enzymes that catalyze ß-lactam hydrolysis. Structural studies of both of these groups of enzymes, their substrates and of ß-lactams have led to the conclusion that ß-lactamases have evolved from a DD-peptidase ancestor. Thus, the active sites of DD-peptidases and serine ß-lactamases are very similar. Why is it then that the active site of a serine ß-lactamase can catalyze hydrolysis of a ß-lactam while that of a DD-peptidase cannot? In view of the active site similarities, why was it necessary for ß-lactamases to evolve at all? The aim of this review is to examine our current understanding of these issues in terms of the crystal structures of the relevant enzymes that are now available, rounding off the analysis with speculation where necessary.

A New Covalent Inhibitor of Class C ß-Lactamases Reveals Extended Active Site Specificity.

O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C ß-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, D-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 10(4) M(-1) s(-1)) of class C ß-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that ß-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent ß-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼ 1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor ß-lactamases.

Interactions of "bora-penicilloates" with serine ß-lactamases and DD-peptidases.

Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial ß-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in ß-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C ß-lactamases but, very unexpectedly, not inhibitors of class A ß-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of ß-lactam antibiotics, where deacylation of ß-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.

Neutral ß-Lactams Inactivate High Molecular Mass Penicillin-Binding Proteins of Class B1, Including PBP2a of MRSA.

The targets of ß-lactam antibiotics are bacterial DD-peptidases (penicillin-binding proteins). ß-Lactam SAR studies over many years have demonstrated the importance of a specifically placed negative charge, usually carboxylate, on these molecules. We show here that neutral analogues of classical ß-lactam antibiotics are of comparable activity to the originals against ß-lactam-resistant high molecular mass DD-peptidases of the B1 class, a group that includes PBP2a of methicillin-resistant Staphylococcus aureus. These neutral ß-lactams may direct new development of antibiotics against certain penicillin-resistant bacteria. These molecules do have antibiotic activity against Gram-positive bacteria.

Kinetics of action of a two-stage pro-inhibitor of serine ß-lactamases.

ß-Lactamase inhibitors are important in medicine in the protection of ß-lactam antibiotics from ß-lactamase-catalyzed destruction. The most effective inhibitors of serine ß-lactamases covalently modify the enzyme active site. We have recently studied O-acyl and O-phosphyl hydroxamates as a new class of such inhibitors. In this paper, we describe our studies of the N-acyl derivatives of a cyclic O-acyl hydroxamic acid, 3H-benzo[d][1,2]oxazine-1,4-dione, and, in particular, the N-tert-butoxycarbonyl derivative. This compound is not a ß-lactamase inhibitor itself but undergoes spontaneous hydrolysis in aqueous solution, yielding an O-phthaloyl hydroxamic acid, which is a ß-lactamase inhibitor. This compound spontaneously, but reversibly, cyclizes in solution to form phthalic anhydride, which is also a ß-lactamase inhibitor. Both inhibitors react to form the same transiently stable phthaloyl-enzyme complex. Thus, we have a two-step cascade, beginning with a pro-inhibitor, in which each step leads to a different inhibitor, presumably with different enzyme specificities. The kinetics of these transformations have been elucidated in detail. The phthaloyl derivatives, where the free carboxylate is important for facile reaction with the enzyme, represent a new lead for serine ß-lactamase inhibitors. Analogues can be conveniently constructed in situ by reaction of nucleophiles with phthalic anhydrides and then screened for activity. Active hits may then become new leads.

Covalent inhibition of serine ß-lactamases by novel hydroxamic acid derivatives.

The effectiveness of ß-lactam antibiotics is greatly limited by the ability of bacteria to produce ß-lactamases. These enzymes catalyze the hydrolysis of ß-lactams and thus loss of their antibiotic activity. The search for inhibitors of ß-lactamases began soon after ß-lactams were introduced into medical practice and continues today. Some time ago, we introduced a new class of covalent serine ß-lactamase inhibitors, the O-aryloxycarbonyl hydroxamates, that inactivated these enzymes by a unique mechanism in which the active site became cross-linked. We describe in this paper some new variants of this class of inhibitor. First, we investigated compounds in which more polar hydroxamates were incorporated. These were generally not more active than the original compounds against representative class A and class C ß-lactamases, but one of them, 1-(benzoyl)-O-(phenoxycarbonyl)-3-hydroxyurea, was significantly more stable in solution, thus revealing a useful platform for further design. Second, we describe a series of O-(arylphosphoryl) hydroxamates that are also irreversible inactivators of class A and class C ß-lactamases, by phosphorylation of the enzyme, as revealed by mass spectra. These compounds did not, however, cross-link the enzyme active site. A striking feature of their structure-activity profile was that hydroxamate remained the leaving group on enzyme phosphorylation rather than aryloxide, even though the aryloxide was intrinsically the better leaving group, as indicated by pKa values and demonstrated by the products of hydrolysis in free solution. Model building suggested that this phenomenon arises from the relative affinity of the enzyme active site components for the two leaving groups. The results obtained for both groups of inhibitors are important for further optimization of these inhibitors.

Dual substrate specificity of Bacillus subtilis PBP4a.

Bacterial dd-peptidases are the targets of the ß-lactam antibiotics. The sharp increase in bacterial resistance toward these antibiotics in recent years has stimulated the search for non-ß-lactam alternatives. The substrates of dd-peptidases are elements of peptidoglycan from bacterial cell walls. Attempts to base dd-peptidase inhibitor design on peptidoglycan structure, however, have not been particularly successful to date because the specific substrates for most of these enzymes are unknown. It is known, however, that the preferred substrates of low-molecular mass (LMM) class B and C dd-peptidases contain the free N-terminus of the relevant peptidoglycan. Two very similar LMMC enzymes, for example, the Actinomadura R39 dd-peptidase and Bacillus subtilis PBP4a, recognize a d-α-aminopimelyl terminus. The peptidoglycan of B. subtilis in the vegetative stage, however, has the N-terminal d-α-aminopimelyl carboxylic acid amidated. The question is, therefore, whether the dd-peptidases of B. subtilis are separately specific to carboxylate or carboxamide or have dual specificity. This paper describes an investigation of this issue with B. subtilis PBP4a. This enzyme was indeed found to have a dual specificity for peptide substrates, both in the acyl donor and in the acyl acceptor sites. In contrast, the R39 dd-peptidase, from an organism in which the peptidoglycan is not amidated, has a strong preference for a terminal carboxylate. It was also found that acyl acceptors, reacting with acyl-enzyme intermediates, were preferentially d-amino acid amides for PBP4a and the corresponding amino acids for the R39 dd-peptidase. Examination of the relevant crystal structures, aided by molecular modeling, suggested that the expansion of specificity in PBP4a accompanies a change of Arg351 in the R39 enzyme and most LMMC dd-peptidases to histidine in PBP4a and its orthologs in other Bacillus sp. This histidine, in neutral form at pH 7, appeared to be able to favorably interact with both carboxylate and carboxamide termini of substrates, in agreement with the kinetic data. It may still be possible, in specific cases, to combat bacteria with new antibiotics based on particular elements of their peptidoglycan structure.

Inhibition of DD-peptidases by a specific trifluoroketone: crystal structure of a complex with the Actinomadura R39 DD-peptidase.

Inhibitors of bacterial DD-peptidases represent potential antibiotics. In the search for alternatives to ß-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with DD-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C DD-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [D-α-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 DD-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective DD-peptidase inhibitors and therefore, perhaps, antibiotics.

Crossover inhibition as an indicator of convergent evolution of enzyme mechanisms: a ß-lactamase and a N-terminal nucleophile hydrolase.

O-Aryloxycarbonyl hydroxamates and 1,3,4-oxathiazol-2-ones have been identified as covalent inhibitors of ß-lactamases and proteasomes, respectively. The products of these inhibition reactions are remarkably similar, involving carbonyl cross-linking of the active sites. We have cross-checked these inhibitors, showing that the former inhibit proteasomes and the latter ß-lactamases, to form the same inactive carbonyl adducts. These results are discussed in terms of similarities of the active site structures and catalytic mechanisms. It is likely that a mechanistic imperative has led to convergent evolution of these enzyme active sites, of a ß-lactam-recognizing enzyme and a N-terminal protease belonging to different amidohydrolase superfamilies.

Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-α-hydroxyglycine ester catalyzed by serine ß-lactamases and DD-peptidases.

The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate (5) is a quite effective substrate of serine ß-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC ß-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 ß-lactamases and the Streptomyces R61 and Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since ß-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the ß-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99 ß-lactamase active site suggests that the α-hydroxyl 5 of may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the ß-lactamase active site, although their effects are not additive; this may also be true for inhibitors.

Inhibition of bacterial DD-peptidases (penicillin-binding proteins) in membranes and in vivo by peptidoglycan-mimetic boronic acids.

The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the ß-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (K(i) < 1 mM) to the low-molecular mass PBPs but not to the high-molecular mass enzymes, both in membrane preparations and in whole cells. In two cases, E. coli PBP2 and PBP5, the dissociation constants obtained were very similar to those obtained with the pure enzymes in homogeneous solution. The boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.

4-quinolones as noncovalent inhibitors of high molecular mass penicillin-binding proteins.

Penicillin-binding proteins (PBPs) are important bacterial enzymes that carry out the final steps of bacterial cell wall assembly. Their DD-transpeptidase activity accomplishes the essential peptide cross-linking step of the cell wall. To date, all attempts to discover effective inhibitors of PBPs, apart from ß-lactams, have not led to new antibiotics. Therefore, the need for new classes of efficient inhibitors of these enzymes remains. Guided by a computational fragment-based docking procedure, carried out on Escherichia coli PBP5, we have designed and synthesized a series of 4-quinolones as potential inhibitors of PBPs. We describe their binding to the PBPs of E. coli and Bacillus subtilis. Notably, these compounds bind quite tightly to the essential high molecular mass PBPs.