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Kinetic parameters are reported for the Bacillus cereus P-lactamase Iand P-lactamase II -catalysed hydrolysis of a series of thirty-seven cephalosporins substituted in the 7-position. These are compared with the second-order rate constants for the hydroxide ion-catalysed hydrolysis of these derivatives. There is no significant dependence of the rate of the base-catalysed hydrolysis upon the nature of the side-chain substituent. For P-lactamase I, kc,,/K, varies over 2 x lo5 but for plactamase II the variation with substituents is only 10. For alkyl substituents, k,,JK, increases with chain length and passes through a maximum, for p-lactamase I this is with the undecyl derivative and for P-lactamase II the octylcephalosporin. For p-lactamase I, but not for P-lactamase II, the tbutylcephalosporin is a very poor substrate. There is no evidence for a significant cavity in either enzyme to host aromatic residues. An ionised carboxylate residue on the side-chain significantly reduces reactivity with P-lactamase I but not p-lactamase II. It is suggested that a carboxy group on P-lactamase I acts as a general catalyst facilitating p-lactam C-N bond fission.The two major classes of p-lactam antibiotics are the penicillins (1) and the cephalosporins (2).t Bacteria which are normally susceptible to these antibiotics may be rendered insusceptible if they can produce P-lactamases, enzymes that catalyse the hydrolysis of the p-lactam.There are many different p-lactamases which have been classified on the basis of substrate profile (the ability to catalyse the hydrolysis of various p-lactam antibiotics e.g. penicillinase or cephalosporinase), on the basis of their sequence or on a mechanistic bask2 For example, the TEM2 P-lactamase from E. coli is very efficient and one molecule of the enzyme can catalyse the hydrolysis of 2 OOO molecules of benzylpenicillin in 1 ~e c o n d . ~ However, this enzyme is more than loo00 less reactive towards some cephalo~porins.~ Conversely, one molecule of the P99 P-lactamase can hydrolyse 2 OOO molecules of cephaloridine in 1 second. One mechanistic class comprises plactamases that are serine enzymes; this is subdivided into classes A and C based on the amino acid sequences.2 The other mechanistic class of p-lactamases is distinguished by the requirement for zinc(r1) ions and a lack of sequence homology to enzymes of the other classes. There are two well characterised but unrelated enzymes of this class B-p-lactamase I1 from B. cereus and p-lactamase L-1 from Pseudonomas maltophilia. RCONH RCONH An example of the systematic nomenclature system for the trivially named cephalosporins, includes 3-[(acetyloxy)methyl]-8-oxo-7-(2-pheny1acetamido)-5-thia-1 -azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid for benzylcephalosporin.

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Rate coefficient data are reported for the hydrolysis of benzyl penicillenic acid over pH range 0.5-7 4. The complex rate-pH profile, containing six changes in acidity dependence, is analysed in terms of the dissociation constants and relative reactivities of the tautomeric species present. The yields of the four known reaction products at various pH's provide quantitative support for the mechanistic interpretation.PENICILLENIC acids (I) have a structure tautomeric with that of penicillins. Although U.V. spectral studies demonstrate that the equilibrium concentration in solution is small, in the presence of a reagent such as a mercury salt which disturbs the equilibrium by reacting with the thiol group, the rate of equilibration can be shown to be relatively r a ~i d . ~? ~ Because of their ease of formation and high reactivity with nucleophiles in isolated systems: penicillenic acids have been proposed as intermediates to account for certain reactions of penicillins, such as the binding to proteins in connection with penicillin allergy2 and the formation of penillic acids (II).5 Fragmented studies indicate that the lifetimes of penicillenic acids as active intermediates in solution are relatively short.4 Under a variety of reaction conditions the compounds have been shown to rapidly hydrolyse to penillic acids (11) 5 penamaldic acids (111) ,ly6 penicilloic acids (IV) ,7 and 4-hydroxymethyleneoxazol-5(4H)-one (V) .5 Some confusion as to the stability has arisen due to the formation of more stable disulphides (VI) which have similar U.V. spectra but differ markedly in reactivity both for hydrolysis and reaction with nucleophiles.8 As no comprehensive hydrolysis investigation has previously been undertaken a study has been made of the hydrolysis of 2-benzylpenicillenic acid in the absence of oxidation over a wide range of experimental conditions to determine the factors influencing the rate of hydrolysis and the relative yields of the various reaction products. EXPERIMENTAL Materials.-Benzylpenicillenic acid (I) (R = PhCH,) 322 nm (log E 4.36, lit.,S 4.42)] and 4-(l-carboxyethylaminomethylene) -2-phenyloxazol-5(4H) -one (XI) [&= 349nm (log E 4.50)] were prepared as described previously.8 Benzylpenillic acid (11) (R = PhCH,) and benzylpenicilloic acid (IV) (R = PhCH,) were prepared from penicillin G as described in the literature.gg10 4-Isopropylidene-2-phenyloxazol-5(4H)-one ( X I ) [Amx. 312 nm (log E 4-54), cf. lit.ll Amax. 310 nm (log E 4-52)] was supplied by Dr. J. Preston of these laboratories and D-pencillamine by Koch-Light Laboratories Ltd. Satisfactory n.m.r. and i.r. analyses

N-Cyanourea is demonstrated as an intermediate in the hydrolysis of dicyanamide which is shown to react as its carbodi-imide tautomer. The hydrolysis of N-cyanourea, dicyanamide, and "'-dimethylcyanoguanidine is specific acid-catalysed. General acid catalysis is demonstrated for the hydrolysis of NN'-dimethylcyanoguanidine and is considered to be specific acid-nucleophilic. Assuming the carbodiimide mechanism also holds for the specific acid-catalysed hydrolysis of cyanamide the tautomeric equilibrium constant for formation of the parent ca+rbodi-imide in water at 25" is estimated to be 0.6 x 1 O-'