RESUMO
The Streptomyces R61 DD-peptidase gene encodes a 26-residue C-terminal extension which is not found in the mature protein. When the gene was expressed in Escherichia coli, the extension was not cleaved and the precursor protein was not enzymatically active. It also reacted with penicillins significantly more slowly than the mature protein. The introduction of a 'stop' codon after that corresponding to the C-terminal residue of the mature protein resulted in the production of an active protein in the periplasm of E. coli.
Assuntos
Carboxipeptidases/metabolismo , Precursores Enzimáticos/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Streptomyces/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Carboxipeptidases/química , Carboxipeptidases/genética , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Escherichia coli/genética , Dados de Sequência Molecular , OligodesoxirribonucleotídeosRESUMO
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.
Assuntos
Ativação Enzimática , Lactamas , Processamento de Sinais Assistido por Computador , beta-Lactamas , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Cefaloridina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Temperatura Alta , Cinética , Microcomputadores , Muramilpentapeptídeo Carboxipeptidase/antagonistas & inibidores , Ácido Penicilânico/farmacologia , Software , Espectrofotometria Ultravioleta , Zinco/metabolismo , Inibidores de beta-LactamasesRESUMO
The 26,000-Mr DD-peptidase of Streptomyces K15 binds one equivalent of thiol reagents as 5,5'-dithiobis-(2-nitrobenzoate) or p-chloromercuribenzoate (pCMB). Derivatization of the DD-peptidase by pCMB decreases the efficacy of the initial binding of the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate to the enzyme (K), the rate of enzyme acylation by the donor (K+2) and the rate of enzyme deacylation (k+3). However, the value of the k+2/k+3 ratio, and therefore the percentage of total enzyme which, at saturating concentrations of the donor, is present as acyl-enzyme at the steady state of the reaction, are not modified. The enzyme's binding sites for pCMB and benzylpenicillin are not mutually exclusive. But, when compared with the native enzyme, the pCMB-derivatized enzyme undergoes acylation by benzylpenicillin with a decreased second-order-rate constant (k+2/K) value and gives rise to a penicilloyl adduct of increased stability. Since the acyl-enzyme mechanism is not annihilated by pCMB derivatization, it is proposed that basically, and like all the other DD-peptidases/penicillin-binding proteins so far characterized, the Streptomyces K15 DD-peptidase is an active-site-serine enzyme.
