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The polymerisation of carbonic anhydrides, a reaction important both as a preparative method for the synthesis of polypeptides, and as an example of a less familiar type of polymerisation, has been studied in detail, using sarcosine carbonic anhydride (I). The propagation reaction has been shown to involve a reversibly formed compound between the carbonic anhydride and the polymer; this compound decomposes by both a unimolecular and a bimolecular route. The equilibrium constant for the formation of the intermediate compound, and both velocity constants for its decomposition have been determined in two solvents, and the activation energies and frequency factors calculated. The molecular weight distribution has been calculated from the kinetic scheme; the distribution is extremely sharp.

The sequence around the thiol group in lens proteins has been investigated. The proteins were converted into their carboxy[(14)C]methyl derivatives and submitted to partial acid hydrolysis, or digested with proteolytic enzymes. Acid hydrolysis of bovine alpha-crystallin gives N-seryl-(S-carboxymethyl)cysteine, Ser-CMCys (Waley, 1965a), but this dipeptide is not obtained from beta-crystallin or gamma-crystallin. Trypsin and chymotrypsin also give different peptides from the three crystallins. The radioactive peptide from alpha-crystallin and chymotrypsin has the sequence Ser-CMCys-Ser-Leu; another peptide, Asp-Leu-Leu-Phe, was also identified. The radioactive peptides obtained from bovine alpha-crystallin are probably also obtained from human alpha-crystallin, and from bovine and human albuminoid (the insoluble lens protein). alpha-Crystallin has been fractionated by chromatography in urea on DEAE-cellulose. Comparison of the fractions by peptide ;mapping', and immunochemically, shows that they fall into two classes. The fraction eluted first differs from the later fractions, but the later fractions resemble each other The first fraction may represent impurities, or it may be a structurally different sub-unit of alpha-crystallin.

In Moraxella (Branhamella) catarrhalis and Moraxella nonliquefaciens strains isolated from clinical specimens in the south of Sweden two variants of beta-lactamase were distinguished by isoelectric focusing (IEF). The BRO-1 (Ravasio type) enzyme was the most common in Branhamella catarrhalis, constituting about 90% of the beta-lactamase found in this species, while the BRO-2 enzyme (1908 type) was as common as BRO-1 in Moraxella nonliquefaciens. The determinants mediating the production of BRO-1 and BRO-2 were both transferable by conjugation. Cell-bound beta-lactamase from reference strains producing BRO-1 and BRO-2 could be solubilized by papain digestion. The isoelectric point of the solubilized enzymes differed distinctly between BRO-1 (pI 6.5) and BRO-2 (pI 6.9). The molecular species of BRO-1 and BRO-2 released by papain digestion were purified by affinity chromatography with phenylboronic acid agarose gel. They had identical molecular weights of approximately 28,000. Their kinetic constants were indistinguishable for a number of substrates and beta-lactamase inhibitors.

Previous chapters have described how β-lactam antibiotics act, and how the β-lactam ring is built up. This chapter describes how the four-membered ring is broken down. This is a topic of chemical and clinical interest: β-lactamases are enzymes that bacteria produce to defend themselves against β-lactam antibiotics.1 Since β-lactamases represent a main cause of antibiotic resistance, they can limit the effectiveness of antibiotics and contribute to the ravages of infectious disease. The mechanism of β-lactamase action represents achallenge with twin goals: (i) to understand how β-lactamases act, and how their action differs from that of the transpeptidases described in chapter 5; and (ii) to exploit this understanding. The exploitation will take the form of improved antimicrobially active β-lactams that are inert to β-lactamases, or improved β-lactamase inhibitors.

A convenient and accurate procedure for determining the kinetic parameter Vmax./Km is described. This avoids the error in the usual method of taking the observed first-order rate constant of an enzymic reaction at low substrate concentration as Vmax./Km. A series of reactions is used in which the initial concentration of substrate is below Km (e.g. from 5% to 50% of Km). Measurements are taken over the same extent of reaction (e.g. 70%) for each member of the series, and treated as if the kinetics were truly first-order. The reciprocal of the observed first-order rate constant is then plotted against the initial concentration of substrate: the reciprocal of the ordinate intercept is Vmax./Km. The procedure, as well as being applicable to simple reactions, is shown to be valid when there is competitive inhibition by the product, or when the reaction is reversible, or when there is competitive or mixed inhibition. The hydrolysis of cephalosporin C by a beta-lactamase from Pseudomonas aeruginosa is used to illustrate the method.

When the Michaelis-Menten equation is obeyed, the rate near the beginning of an enzyme-catalyzed reaction (or of an experiment on transport) can be found accurately from the slope of a chord joining two points on the progress curve. This slope gives the rate at an intermediate concentration. Exact values of this intermediate concentration are easily calculated from equations in the text, and a number of values have also been tabulated. Methods of using two chords to find the initial rate are given. A mid-point formula for numerical differentiation is advocated when the Michaelis-Menten equation does not hold.

There are two extracellular β-lactamases produced by Bacillus cereus 569. One of these enzymes, β-lactamase I, is inactivated by 6-β-bromopenicillanic acid: the site of reaction is serine-44. This is a conserved amino acid residue in the other β-lactamases whose structures have been determined, and it becomes a good candidate for an active-site group in these enzymes. The inactivation may involve a rearrangement leading to a dihydrothiazine. The other extracellular enzyme produced by B. cereus , β- lactamase II, is exceptional in requiring metal ions for activity. The Zn II and Co II enzymes (the former is more active) have been studied by nuclear magnetic resonance, and by absorption spectroscopy. The groups that bind the metal ion required for activity are three histidine residues and the enzyme’s sole thiol group.

In summary, Table XVI shows the inhibition profiles of representative beta-lactamases from each major class of Richmond and Sykes. Either resistance (R) or sensitivity (S) is given as a general guide to the type of compounds likely to inhibit each class. Thus the (qualitative) statements regarding the effectiveness of clavulanic acid can be taken to represent those for the penam sulfones and similarly for MM4550 and the other olivanic acids, carpetimycins, PS series, and asparenomycins. This can also be said of cloxacillin and the other aromatic carboxamido penicillins. Compounds are also included which are specifically or particularly inhibitory to certain beta-lactamases.

The conformational motility of beta-lactamase I from Bacillus cereus was studied by hydrogen exhange. The time course of the isotopic replacement of peptide hydrogen atoms was followed by ‘exchange-in’ or ‘exchange-out’ experiments. Many of the substrates for this enzyme that have o-substituted aromatic or heterocyclic side chains (e.g. methicillin or cloxacillin) are known to effect a decrease in enzymic activity (‘substrate-induced deactivation’). There was a marked discontinuity in the exchange-out curve when methicillin or cloxacillin was diffused into the enzyme solution. About one-half of the hydrogen atoms that were probed were affected by the presence of these substrates, and the change in the reactivity of the hydrogen atoms was also large. Substrates that do not bring about deactivation (benzylpenicillin and cephalosporin C) do not affect the hydrogen exchange, nor do reversible competitive inhibitors such as the penicilloic acid or penilloic acid. On the other hand, Zn2+ ions do affect the hydrogen exchange; their effect is similar to that of methicillin or cloxacillin.

beta-Lactamases, enzymes that catalyse the hydrolysis of the beta-lactam ring in beta-lactam antibiotics, are divided into three classes, A, B and C, on the basis of the structures so far determined. There are relatively few effective inhibitors of class C beta-lactamases. A beta-lactam sulphone with a hydroxybenzyl side chain, namely (1′R,6R)-6-(1′-hydroxy)benzylpenicillanic acid SS-dioxide (I), has now been studied. The sulphone is a good mechanism-based inhibitor of class C beta-lactamases. At pH8, the inhibition of a Pseudomonas beta-lactamase is irreversible, and proceeds at a rate that is about one-tenth the rate of concurrent hydrolysis. The labelled enzyme has enhanced u.v. absorption and is probably an enamine. At a lower pH, however, inhibition is transitory.

The binding of ligands to chicken muscle triose phosphate isomerase was studied. Changes in u.v. absorbance of the enzyme were used to measure binding, and the dissociation constant was determined over a range of pH values. The ligands were 2-phosphoglycollate and rac-glycerol 3-phosphate (only the D-isomer, sn-glycerol 1-phosphate, binds appreciably). Non-linear regression was used to fit calculated curves to the experimental points and hence to compare different models. Both active sites in the dimeric enzyme probably bound 2-phosphoglycollate, without any interaction between the sites. The results of crystallographic analysis [phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc Trans. 5, 642–647], and experiments on the 1H, 13C and 31P n.m.r. of enzyme or 2-phosphoglycollate were combined with the present results to provide the basis for a model in which binding depends on glutamic acid-165 being protonated and on the ligant being fully ionized; additionally, binding affects the ionization of one histidine residue (probably histidine-100). The binding of the glycerol 3-phosphate, on the other hand, was independent of pH over the range pH 6.5–8.5 but decreased at lower pH values. This is explained on a model in which the binding of the monoanion of the ligand is markedly affected by the protonation of a residue in the enzyme, but the binding of the dianion is only slightly affected by this ionization.

In the presence of a reporter substrate, the progressive inactivation of an enzyme was easily studied by directly transmitting absorbance readings to a microcomputer. Pseudo-first order rate constants as high as 0.3 sec-1 were rapidly and accurately measured. When utilization of the reporter substrate did not exceed 10%, the rate of the reaction (vt) could be considered as proportional to the active enzyme concentration at any time during the analysis and the decrease of vt was first order with time. This simple method was used to follow the inactivation of beta-lactamases (EC 3.5.2.6) by various physical and chemical agents. When a large proportion (30-80%) of reporter substrate was destroyed, a correction was introduced to account for the corresponding decrease of its rate of utilization. This enabled experiments to be performed with a DD-peptidase and a substrate exhibiting a low delta epsilon upon hydrolysis. For the first time, the inactivation of a penicillin-sensitive enzyme by a beta-lactam could be continuously and directly observed. Finally, the method was extended to the study of hysteresis phenomena.

The kinetics of the hydrolysis of two cephalosporins by β-lactamase I from Bacillus cereus 569/H/9 has been studied by single-turnover and steady-state methods. Single-turnover kinetics could be measured over the time scale of minutes when cephalosporin C was the substrate. The other substrate, 7-(2′,4′-dinitrophenylamino)deacetoxycephalosporanic acid, was hydrolysed even more slowly, and has potential for use in crystallographic studies of β-lactamases. Comparison of single-turnover and steady-state kinetics showed that, for both substrates, opening the β-lactam ring (i.e. acylation of the enzyme) was the rate-determining step. Thus the non-covalent enzyme-substrate complex is expected to be the intermediate observed crystallographically.

1. The Zn(II)-requiring beta-lactamase from Bacillus cereus 569/H/9, which has two zinc-binding sites, was examined by 270 MHz 1H n.m.r. spectroscopy. Resonances were assigned to five histidine residues. 2. Resonances attributed to three of the histidine residues in the apoenzyme shift on the addition of one equivalent of Zn(II). 3. Although these three histidine residues are free to titrate in the apoenzyme, none of them titrates over the pH range 6.0–9.0 in the mono-zinc enzyme. 4. The ability of the C-2 protons of these three histidine residues to exchange with solvent (2H2O) is markedly decreased on Zn(II) binding. 5. It is proposed that these three histidine residues act as zinc ligands at the tighter zinc-binding site. 6. Resonances attributed to a fourth histidine residue shift on addition of further zinc to the mono-zinc enzyme. It is proposed that this histidine residue acts as a Zn(II) ligand at the second zinc-binding site.

Many clinically important or mechanistically interesting inhibitors react with enzymes by a branched pathway in which inactivation of the enzyme and formation of product are competing reactions. The steady-state kinetics for this pathway [Waley (1980) Biochem. J. 185, 771-773] gave equations for progress curves that were cumbersome. A convenient linear plot is now described. The time (t1/2) for 50% inactivation of the enzyme (this is also the time for 50% formation of product), or for 50% loss of substrate, is measured in a series of experiments in which the concentration of inhibitor, [I]0, is varied; in these experiments the ratio of the concentration of enzyme to the concentration of inhibitor is kept fixed. Then a plot of [I]0 X t1/2 against [I]0 is linear, and the kinetic parameters can be found from the slope and intercept. Furthermore, simplifications of the equations for progress curves are described that are valid when the concentration of inhibitors is high, or is low, or when the extent of reaction is low. The use of simulated data has shown that the recommended methods are not unduly sensitive to experimental error.

The polymerization of carbonic anhydrides, a reaction important but>u as a preparative method for the synthesis of polypeptides and as an example of a less familiar type of polymerization, has been studied in detail using sarcosine carbonic anhydride (I). The propagation reaction has been shown to involve a reversibly formed compound between the carbonic anhydride and the polymer; this compound decomposes by both a unimolecular and a bimolecular route. The equilibrium constant for the formation of the intermediate compound, and both velocity constants for its decomposition have been determined in two solvents, and the energies of activation and frequency factors calculated. The molecular weight distribution has been calculated; it is extremely sharp. The viscosities of the polymers prepared by this reaction have been measured, and the relation between the intrinsic viscosity and the molecular weight established.

The inhibition constant Ki in the common case of competitive inhibition can be obtained by simple comparison of progress curves in the presence and in the absence of inhibitor. The difference between the times taken for the concentration of substrate to fall to the same value is used to obtain Ki. The procedure to use when the product inhibits is described. When there is mixed inhibition, reactions at different substrate concentrations are used to obtain both inhibition constants.

The use of two substrates, one of them a reporter substrate, is often convenient. The time course of an enzymic reaction with competing substrates is given explicitly in the present paper. Kinetic parameters can be readily obtained. The method is applied to the hydrolysis of benzylpenicillin, with cephalosporin C as the reporter substrate, catalysed by a Pseudomonas beta-lactamase.

beta-Lactam antibiotics--the penicillins, cephalosporins and related compounds--act by inhibiting enzymes that catalyse the final stages of the synthesis of bacterial cell walls. Recent crystallographic studies of representative enzymes are beginning to reveal the structural bases of antibiotic specificity and mechanism of action, while intensive efforts are being made to understand the beta-lactamase enzymes that are largely responsible for bacterial resistance to these antibiotics. It has been suggested that the beta-lactamases and beta-lactam target enzymes may be evolutionarily related and some similarity of amino-acid sequence around a common active-site serine residue supports this idea. We present here the first evidence from a comparison of three-dimensional structures in support of this hypothesis: the structure of beta-lactamase I from Bacillus cereus is similar to that of the penicillin-sensitive D-alanyl-D-alanine carboxypeptidase-transpeptidase from Streptomyces R61.

Structure of chicken muscle triose phosphate isomerase determined crystallographically at 2.5A resolution: using amino acid sequence data

β-Lactamases continue to play an important part in antibiotic resistance in bacteria (see, for example, Medeiros, 1984). The prevalence of β-lactamases thus concerns clinicians, and stimulates pharmaceutical companies to produce (supposedly) β-lactamaseresistant drugs. β-Lactamases appear to be as diverse as they are efficient, and one of the reasons for studying their mechanisms is to bring order into their classification. This has been successful, in that a simple binary classification may apply widely. It is perhaps worth mentioning that most of the detailed knowledge of β-lactamase mechanisms dates from 1978. Thus from the point of view of their mechanisms, we can now divide those β-lactamases (or rather, those that have been studied in enough depth) into ‘serine’ enzymes and ‘metal’ enzymes. A second division splits the ‘serine’ enzymes into two classes (called class A and class C), based on their amino acid sequences (Ambler, 1980; Jaurin & Grundstrom, 1981).

In the presence of a reporter substrate, the progressive inactivation of an enzyme was easily studied by directly transmitting absorbance readings to a microcomputer. Pseudo-first order rate constants as high as 0.3 sec-1 were rapidly and accurately measured. When utilization of the reporter substrate did not exceed 10%, the rate of the reaction (vt) could be considered as proportional to the active enzyme concentration at any time during the analysis and the decrease of vt was first order with time. This simple method was used to follow the inactivation of beta-lactamases (EC 3.5.2.6) by various physical and chemical agents. When a large proportion (30-80%) of reporter substrate was destroyed, a correction was introduced to account for the corresponding decrease of its rate of utilization. This enabled experiments to be performed with a DD-peptidase and a substrate exhibiting a low delta epsilon upon hydrolysis. For the first time, the inactivation of a penicillin-sensitive enzyme by a beta-lactam could be continuously and directly observed. Finally, the method was extended to the study of hysteresis phenomena.

The effectiveness of beta-lactam antibiotics against Gram-negative bacteria that contain beta-lactamases is considered in terms of three factors. These are a rate constant for reaction of the beta-lactam antibiotic with a transpeptidase, the kinetic parameters for hydrolysis of the antibiotic by the beta-lactamase, and the permeability of the outer membrane. The concept of a dimensionless permeability number (Pn) is developed. When the permeability number is much less than one then permeability is important, and the MIC may be raised by a factor of up to 1/Pn. When the antibiotic is sufficiently reactive its effectiveness is given by the 'reactivity-permeability' product.

The 1H n.m.r. spectra of beta-lactamase II in the presence of Co(II) were studied. Analysis of the spectra suggests that Co(II) binds at the same two metal-binding sites as does Zn(II). The binding of Co(II) at the first site is much weaker than the binding of Zn(II) at this site, whereas the binding of Co(II) at the second site is tighter than the binding of Zn(II). The binding of Co(II) to the mono-zinc(II)-enzyme caused only one marked change in the spectrum, namely a decrease in the intensity of the resonances assigned to the C-2 and C-4 protons of one histidine residue (residue E). However, when the spectra of the apoenzyme and the Co(II)-enzyme were compared, there were many differences. A significant fraction of the protons in the whole molecule are affected by the binding of Co(II) at the first metal-ion-binding site (where the ligands are the enzyme's sole thiol group and three histidine residues). This may be because the first site is internal, or because of a difference in conformation between the apoenzyme and the mono-Co(II)-enzyme. The second site may be located on the surface of the molecule.

The inactivation of beta-lactamase I by preparations of 6alpha-bromopenicillanic acid showed unexpected kinetic features that indicated that the active species was the 6beta-epimer. Samples containing 6beta-bromopenicillanic acid have been synthesized and shown to inactivate the enzyme in a rapid stoicheiometric reaction.

Reversible competitive inhibitors of a penicillinase, beta-lactamase 1 from Bacillus cereus, were studied. These represent the first inhibitors of a penicillinase that lack the beta-lactam ring. The products of the enzymic reaction, namely penicilloic acids, are inhibitors; their decarboxylation products, the penilloic acids, are also inhibitors, and have somewhat lower Ki values. Inhibitors have been prepared from benzylpenicillin, phenoxymethyl-penicillin, methicillin (2,6-dimethoxybenzamidopenicillanic acid) and 3-hydroxy-4-nitrobenzamidopenicillanic acid. Decarboxylation of the penicilloic acids from benzyl-penicillin, or from phenoxymethylpenicillin, leads to epimerization (at C-5) of the penilloic acid. Nuclear-magnetic resonance spectroscopy at a frequency of 270 MHz can distinguish the epimers. Other competitive inhibitors studied were boric acid, benzene boronic acid and m-aminobenzeneboronic acid. Boric acid itself was the best inhibitor of beta-lactamase I so far found.

LOOMIS1 has shown that the ‘feeding reaction’ which Hydra littoralis exhibit after they have captured living prey is controlled chemically: the feeding reaction is elicited by glutathione released from the prey. In the absence of prey, glutathione induces writhing of the tentacles and, in higher concentrations, glutathione causes the hydra to retract the tentacles. These effects were found to be more marked when the hydra had previously been starved for several days. Loomis found that the effect was quite specific and that even the closely related aspartathione was inactive. Two analogues of glutathione (ophthalmic acid and norophthalmic acid) have recently been isolated from calf lens2–4; they differ structurally from glutathione in that the SH group is replaced by a CH3group in ophthalmic acid, and an H atom in norophthalmic acid. We have now found that these analogues of glutathione also elicit the feeding reactions in Hydra littoralis.

GLYCIDOL (2,3-epoxypropanol) phosphate inactivates and becomes covalently bound to triose phosphate isomerase (EC 5.3.1.1; TIM); the kinetics and stoichiometry of the reaction, and the structure of the reagent, indicate a specific reaction at the active centre of the enzyme1. The site that is labelled by this inhibitor, and by glycidol in the presence of inorganic phosphate, has now been identified, lodohydroxyacetone phosphate2 and bromohydroxyacetone phosphate3 are also specific inhibitors of TIM. The amino-acid sequence round the site in chicken muscle TIM labelled by the bromo compound has been established4; we were greatly helped by knowing the sequence of their peptide.

1. alpha-Crystallin has been isolated from the cortex of ox lens by isoelectric precipitation followed by chromatography on DEAE-cellulose. The amino acid composition is in agreement with that reported for alpha-crystallin prepared by a different method. There is one thiol group/20000g. of protein (20000 is the order of magnitude of the sub-unit molecular weight), and disulphide bonds are absent. 2. The thiol group has been alkylated with radioactive iodoacetate in the presence of urea. 3. Partial acid hydrolysis of the alkylated protein gives, according to the conditions, mainly three radioactive peptides or nearly exclusively one radioactive dipeptide. The dipeptide is N-seryl-(S-carboxymethyl)cysteine, Ser-CMCys. The two other peptides are probably the tripeptides related to Ser-CMCys. 4. The simplest interpretation of these results is that the sequence around the cysteine residue is a common structural feature of the sub-units of alpha-crystallin.

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified.