Kinetics of enzyme acylation and deacylation in the penicillin acylase‐catalyzed synthesis of β‐lactam antibiotics

Abstract

Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic b-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 acylenzyme. 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 k cat 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 k cat of NIPAOB resulted from an inverse isotope effect at low concentrations of D 2 O, which is overridden by a large normal isotope effect at large molar fractions of D 2 O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D 2 O compared to H 2 O. Deacylation of the acyl-enzyme was studied by nucleophile competition and inhibition experiments. The b-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 steadystate, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of b-lactam antibiotics were obtained.Keywords: penicillin acylase; antibiotic synthesis; kinetic mechanism; kinetic isotope effect.Penicillin acylase (PA) (EC 3.5.1.11) of Escherichia coli ATCC 11105 hydrolyses penicillin G to produce phenylacetic acid and 6-aminopenicillanic acid (6-APA). The latter compound can be used industrially as a precursor for the synthesis of semisynthetic b-lactam antibiotics. In synthetic reactions, 6-APA is coupled to an acyl group in a kinetically controlled conversion in which the acyl group is supplied as an activated precursor, e.g. an ester or an amide [1]. This condensation reaction can also be catalysed by PA and the yield of the desired product is strongly dependent on the mechanism and kinetic properties of the catalyst [2].The catalytic mechanism of PA involves nucleophilic attack of the active-site serine, bS1, on the carbonyl carbon of the amide or ester bond of the substrate [3] (residues are labelled to indicate the subunit (a or b) and their position in the subunit). Via a tetrahedral intermediate that is stabilized by hyd...

Penicillin G acylase as chiral selector in LC and CE: Exploring the origins of enantioselectivity☆

“…The active site is at the bottom of a conic depression formed by residues of the two sub-units, which are tightly intertwined. The crystal structure of the enzyme complexed with different side chain ligands has been reported in different papers [33,[35][36][37][38][39].…”

Improved β-lactam acylases and their use as industrial biocatalysts

“…In the past year a kinetic analysis of the penicillin reaction steps was published with emphasis not only on the hydrolysis reaction, but also on the synthetic reaction [32]. The acylation step and the deacylation step have been described as separate steps in the reaction and it was shown that the breakdown of the acyl-enzyme is much faster than its formation, implying that the rate of hydrolysis is set entirely by the acylation step.…”

Hydrolysis of phenylacetanilides catalyzed by penicillin G acylase from Alcaligenes faecalis: Sensitivity of the reaction to substitution in the leaving group

“…However, the subtle differences in the structure of the enzyme and enzyme-substrate interactions in the active site can strongly affect the energetics of the reaction and have a major impact on the efficiency of catalysis [1,13,14]. Penicillin G acylase from Alcaligenes faecalis (Af-PGA) is structurally very similar to the Escherichia coli enzyme (Ec-PGA) [11].…”