Alkema Wynand B L, de Vries Erik, Floris René, Janssen Dick B
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, the Netherlands.
Eur J Biochem. 2003 Sep;270(18):3675-83. doi: 10.1046/j.1432-1033.2003.03728.x.
Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic beta-lactam antibiotics via formation of a covalent acyl-enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped-flow experiments with the penicillin and ampicillin analogues 2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and d-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) showed that the rate-limiting step in the conversion of penicillin G and ampicillin is the formation of the acyl-enzyme. The phenylacetyl- and phenylglycyl-enzymes are hydrolysed with rate constants of at least 1000 s-1 and 75 s-1, respectively. A normal solvent deuterium kinetic isotope effect (KIE) of 2 on the hydrolysis of 2-nitro-5-[(phenylacetyl)amino]-benzoic acid (NIPAB), NIPGB and NIPAOB indicated that the formation of the acyl-enzyme proceeds via a general acid-base mechanism. In agreement with such a mechanism, the proton inventory of the kcat for NIPAB showed that one proton, with a fractionation factor of 0.5, is transferred in the transition state of the rate-limiting step. The overall KIE of 2 for the kcat of NIPAOB resulted from an inverse isotope effect at low concentrations of D2O, which is overridden by a large normal isotope effect at large molar fractions of D2O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D2O compared to H2O. Deacylation of the acyl-enzyme was studied by nucleophile competition and inhibition experiments. The beta-lactam compound 7-aminodesacetoxycephalosporanic acid (7-ADCA) was a better nucleophile than 6-aminopenicillanic acid, caused by a higher affinity of the enzyme for 7-ADCA and complete suppression of hydrolysis of the acyl-enzyme upon binding of 7-ADCA. By combining the results of the steady-state, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of beta-lactam antibiotics were obtained.
青霉素酰化酶通过形成共价酰基 - 酶中间体催化半合成β - 内酰胺抗生素的水解和合成。研究了这些反应的动力学和机理方面。用青霉素和氨苄青霉素类似物2 - 硝基 - 5 - 苯乙酰氧基 - 苯甲酸（NIPAOB）和d - 2 - 硝基 - 5 - [（苯甘氨酰基）氨基] - 苯甲酸（NIPGB）进行的停流实验表明，青霉素G和氨苄青霉素转化过程中的限速步骤是酰基 - 酶的形成。苯乙酰基 - 酶和苯甘氨酰基 - 酶的水解速率常数分别至少为1000 s⁻¹和75 s⁻¹。2 - 硝基 - 5 - [（苯乙酰基）氨基] - 苯甲酸（NIPAB）、NIPGB和NIPAOB水解时正常的溶剂氘动力学同位素效应（KIE）为2，表明酰基 - 酶的形成通过一般酸碱机制进行。与这种机制一致，NIPAB的kcat的质子丰度表明，在限速步骤的过渡态中有一个质子以0.5的分馏系数转移。NIPAOB的kcat的整体KIE为2，这是由于在低浓度D₂O时的逆同位素效应，而在高摩尔分数的D₂O时被大的正常同位素效应所掩盖。在甘油存在下的速率测量表明，逆同位素效应源于D₂O与H₂O相比更高的粘度。通过亲核试剂竞争和抑制实验研究了酰基 - 酶的脱酰化作用。β - 内酰胺化合物7 - 氨基去乙酰氧基头孢烷酸（7 - ADCA）是比6 - 氨基青霉烷酸更好的亲核试剂，这是由于酶对7 - ADCA的亲和力更高，并且7 - ADCA结合后酰基 - 酶的水解被完全抑制。通过结合稳态、预稳态和亲核试剂结合实验的结果，获得了β - 内酰胺抗生素合成和水解的相关动力学常数的值。
