Interaction between the Exocellular DD‐Carboxypeptidase‐Transpeptidase from Streptomyces R61, Substrate and β‐Lactam Antibiotics

Abstract

The interaction between the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61 and p-lactam antibiotics is a multistep process during which a rather stable enzyme . antibiotic complex is formed. This mechanism of interaction is compatible with Lineweaver-Burk plots that are typical of a competitive inhibition of the hydrolysis of the peptide donor by the antibiotic. In fact, however, the same Lineweaver-Burk plots can be obtained on the basis of a non-competitive type of inhibition. At present, a choice between the two models cannot be made.Recent studies showed that the interaction between the exocellular DD-carboxypeptidase-transpeptidase from Streptomvces R61 (E) and p-lactam antibiotics (I) is a multistep reaction [I]. Once formed, the equimolar and inactive complexes EI give rise to modified complexes EI* which, in turn, undergo breakdown with the concomitant release of the reactivated enzyme (E) and of a modified and inactive antibiotic molecule (X). Depending upon the antibiotic, the EI* complexes have at 37 "C long half-lives of 40 -11 000 min. The rates of formation of complexes EI*, as determined by measuring the quenching of the fluorescence of the R61 enzyme in the presence of large amounts of antibiotics, suggested that the simplest mechanism which accounted for all the experimental results so far accumulated, was :where the formation of complex EI was a rapid equilibrium process [I]. Earlier studies [2] had shown that by incubating together the R61 enzyme, the tripeptide AC,-L-LYS-D-Ala-D-Ala donor and a p-lactam antibiotic for some time (30 -60 min) at 37 "C and on the basis of the residual activity measured under these conditions, Lineweaver-Burk plots were obtained according to which the R61 enzyme was competitively inhibited by the antibiotics with regard to the peptide donor Eur. J. Biochem. 57 (1975) (DD-carboxypeptidase activity: Ac,-L-Lys-D-Ala-D-The enzyme used in these experiments had been purified to protein homogeneity; the antibiotic concentrations were much higher than that of the enzyme and the amount of hydrolysis product formed never exceeded 15% of the original amount of substrate. Because of this low utilization of substrate, it was assumed that the experiments had been carried out under conditions of initial velocity. On the basis of the model proposed above for the interaction between the enzyme and penicillin, however, it appears that this assumption was not justified. Indeed, the estimation of the velocity of the formation of complex EI* showed that the concentration of free enzyme and hence the velocity of the reaction continuously decreased throughout the incubation of the reaction mixture. It thus became essential to examine whether or not the 'competitive' inhibition (in Lineweaver-Burk plots) was compatible with the mechanisms proposed for the interaction between the R61 enzyme and penicillin. THEORETICAL BACKGROUND SymbolsThe symbols are those used previously [I]. In addition, S = substrate (Ac,-L-Lys-D-Ala-D-Ala) ; [S] = substrate concentration; P = ...

Mutations in ponA , the Gene Encoding Penicillin-Binding Protein 1, and a Novel Locus, penC , Are Required for High-Level Chromosomally Mediated Penicillin Resistance in Neisseria gonorrhoeae

“…where E is the enzyme, S is a ␤-lactam antibiotic, and P is the inactive degradation product (13,22). The effectiveness of an antibiotic is defined by two parameters: (i) the second-order specificity constant, k 2 /KЈ, where KЈ ϭ k Ϫ1 /k ϩ1 ; and (ii) the rate constant for hydrolysis of the acyl-enzyme complex, k 3 .…”

Use of Model Enzymes in the Determination of the Mode of Action of Penicillins and Delta3-Cephalosporins

“…In many cases, kinetics suggest a competitive inhibition. However, because of the very rapid forma tion of a stable complex EI*, the concentration of EL and of EID, may be so small that a seemingly competitive inhibition may be obtained even if the interaction is truly noncompetitive (83 of the L-form of Proteus, in spite of a very rapid formation of complex EI * (k31 K = 2-8 X 104M-I sec-I) benzylpenicillin inhibits the hydrolysis of UDP-N-acetylmuramyl-pentapeptide in a noncompetitive manner, with a dissociation constant K' K' EI +D �E ID lower than 3 fLM (67). The interactions between the R61 enzyme and 6-aminopenicillanic acid or between the G enzyme and cephalosporin C are characterized by a very low k3 value (l� sec -I; Table I) so that the inhibition of the enzyme activity by the ,B-Iactams can be studied under conditions where it is due almost exclusively to the fo rmation of complex EL since complex EI * is virtually not fo rmed.…”

Crystal Structure of a Deacylation-defective Mutant of Penicillin-binding Protein 5 at 2.3-Å Resolution

“…These calculations showed that two derivatives, mercury cyanide (HgCN 2 ) and di--iodobis-(ethylenediamine) di-platinum nitrate (PIP), were of the best quality and sufficient to solve the structure. All calculations to this point were performed using PHASES (25). At this stage the derivative and native data were merged using SCALEIT (26), and the final phasing calculation was performed using SHARP (27) and included anomalous data.…”