Studies on enzyme models

Abstract

The thesis is subdivided into three main chapters. The first chapter deals with a general introduction on the mechanism of enzyme action and the study of enzyme models mimicking some enzymes. The second chapter consists of a formal introduction to the present work, the design and building of model systems simulating the action of penicillin acylase for the specific hydrolysis of benzylpenicillin to 6?aminopenicillanic acid (6?APA). Section A deals with the syntheses of simple model catalysts and Section B with those of more complex model catalysts. The third chapter deals with the assessment of thermodynamic energy parameters of association of catalysts with the substrate penicillin?G and the kinetics of the hydrolytic process.

Chapter I – General Introduction In this chapter, several concepts of Koshland and Neet such as orientation effect, proximity effect, acid–base catalysis, charge?relay systems, and orbital steering effect are discussed. These contribute either singly or cumulatively to the rate acceleration process. Some aspects of these parameters contributing to substrate specificity of enzymes and models are reviewed in a general manner.

Chapter II – Model Catalysts for Penicillin Hydrolysis The enzymatic and chemical deacylation of penicillin?G to 6?APA are discussed. The design parameters of an organic catalyst to simulate the action of penicillin acylase are considered with respect to two components:

The catalytic site

The affinity site

For the catalyst group, bifunctional reagents such as hydroxylamine, hydrazine, guanidine, imidazole, 2?hydroxypyridine, and several other systems were chosen. To attach these bifunctional catalyst groups specifically to the target bond, the following structural features of benzylpenicillin were taken into account:

The hydrophobic benzyl group

The target bond (6?amide linkage)

Polar points: carbonyl oxygen, carbon, nitrogen, and sulfur in the ??lactam thiazolidine ring

The hydrophobic isobutyl group in the thiazolidine ring

The polar carboxylate anion at the end of the molecule

In Section A, simple catalysts were synthesized for binding to the benzyl group (hydrophobic site?1) of penicillin through hydrophobic associations. All these catalysts had a benzyl group attached to associate by hydrophobic interactions with the benzyl group of penicillin?G in aqueous systems.

In Section B, two complex catalysts were synthesized, having a benzyl group connected at one end to hydroxylamine and a 9–10 carbon side chain at the other end containing a strategically located quaternary ammonium group juxtaposed to an isobutyl residue to bind with the thiazolidine end of penicillin.

Chapter III – Thermodynamic and Kinetic Studies This chapter describes the spectral methods used to calculate thermodynamic parameters for the association of catalysts with penicillin?G. By studying the increase in optical density of a charge?transfer band of the catalyst (291 nm) with graded concentrations of penicillin?G, it was possible to estimate the equilibrium constant and free energy of the association process.

The assignment of the 291 nm band to charge?transfer was made from several considerations:

This transition could be observed in dibenzylhydroxylamine in aqueous solution but not in non?polar solvents.

The band was initially very feeble in complex catalysts but was enhanced dramatically on addition of benzylpenicillin.

It was possible to isolate a Michaelis?type 1:1 complex between dibenzylhydroxylamine and penicillin?G. NMR data of this complex showed a downfield shift of the aromatic protons by 10 Hz due to lamination of the phenyl rings. The association of dibenzylhydroxylamine had an equilibrium constant of 9800, corresponding to ?G of –5.5 kcal/mol. The associative process had a positive ?S of 1.78 kcal, indicating that entropy?driven hydrophobic association was the main factor in Michaelis complex formation.

The bis p?nitrobenzylhydroxylamine gave a minimum ?G of association of –7.2 kcal. In this case, ?H became negligible (4–25 cal). Both catalysts bound penicillin with approximately the same entropy, although hydrolysis to 6?APA was faster by a factor of four in the bis p?nitrobenzylhydroxylamine system. The complex catalyst showed a lower ?G of binding (–5.1 kcal), presumably because hydrophobic association energy of phenyl rings was utilized to break micellar associations of the catalyst chains.

Kinetic studies were conducted in detail with dibenzylhydroxylamine. The pH?rate profile showed a typical bell?shaped optimum at pH 6. Rates increased with temperature up to ~50?°C, but side reactions and product decomposition limited experiments. At this temperature, the first?order rate constant of 6?APA formation from penicillin by dibenzylhydroxylamine was ~2.45 × 10?? min?¹. Complex catalysts were marginally better, with k ~5.1 × 10?? min?¹. The bis p?nitrobenzylhydroxylamine had a rate of 9.2 × 10?? min?¹, and polymeric dibenzylhydroxylamine ~8 × 10?? min?¹.

All other catalysts were less efficient than dibenzylhydroxylamine. In terms of rates, the enzyme from B. megaterium is 1.6 × 10? times faster on a molar basis and ~2700 times faster on a weight basis than dibenzylhydroxylamine.

All catalysts suffered severe product inhibition, stopping hydrolysis at 12–16% 6?APA yield. The dibenzylhydroxylamine–penicillin system showed competitive inhibition with phenylacetic acid, while complex catalysts were inhibited by both phenylacetic acid and 6?APA.

The chapter concludes with implications of these data, both in terms of specificity and the mechanism of the hydrolytic reaction.