Studies on enzyme models

Abstract

The thesis entitled “Studies on Enzyme Models” is subdivided into two parts. The first part deals with a model system consisting of vanadium pentoxide and hydrogen peroxide, simulating the action of a fungal hydroxylase from A. niger NCIM 612. The second part involves designing a suitable metal?complex model for mimicking the action of penicillin acylase for the specific hydrolysis of benzylpenicillin to 6?aminopenicillanic acid (6?APA). The first part, dealing with fungal hydroxylase, is divided into three sections:

Section 1.1 consists of a general introduction to the mechanism of biological oxygenation and a survey of enzyme?model systems, starting from Fenton’s reagent and the Udenfriend system to various cytochrome P?450 models. This section also states the objectives of the present work. Section 1.2 describes the materials and methods employed in the vanadium?pentoxide–hydrogen?peroxide system and the fungal system. Section 1.3 discusses the results obtained from both systems.

Section 1.1 This section reviews:

Enzymes involved in oxygenation Model systems for the hydroxylation of organic substrates, including Fenton’s reagent, the Udenfriend modification, the Hamilton system, flavin?dependent and pteridin?dependent hydroxylations Hydroxylations mediated by peroxidase (horse?radish peroxidase) and peroxy acids Cytochrome P?450 and various model systems simulating aspects of P?450 oxygen?transfer reactions

Model systems involving iron–thiol, hemin–thiol complexes, chromyl reagents, and Fe²?/O?–methanol systems are also discussed. Finally, hydroxylation of cyclohexene to cyclohexanol using Mn(III)–porphyrin–O?–NaBH? is summarised.

Section 1.2 This section describes the oxidation of various monoterpenoids and alicyclic systems using the polyperoxyvanadate (PPV) system. The study shows:

??Pinene ? (+)?trans?sobrerol, (+)?trans?verbenol, (+)?verbenone ??Pinene ? (+)?trans?pinocarveol, (–)?pinocarvone, (+)?myrtenol d?Limonene ? (–)?trans?carveol, (+)?carvone, (+)???terpineol Cyclohexene ? (+)?cyclohex?2?en?1?ol, cyclohex?2?en?1?one, (+)?cis?cyclohex?3?en?1,2?diol 1?Methyl-, 3?methyl-, and 4?methylcyclohexenes ? allylic alcohols and ketones at all possible positions, plus rearranged products 3?Methylcyclohexene ? 1?methylcyclohex?2?en?1?ol (major), 4?methylcyclohex?2?en?1?one 4,4?Dimethylcyclohexene (fungal hydroxylation) ? 4,4?dimethylcyclohexanol (major), 5,5?dimethylcyclohex?2?en?1?ol, 5,5?dimethylcyclohex?2?en?1?one

Earlier fungal oxygenation data showed product patterns similar to PPV but with cis stereochemistry; PPV yielded mostly trans products.

Section 1.3 This section discusses the oxygenating species involved in hydroxylation—oxygen, perhydroxyl radical, peroxide, hydroxyl radical, and OH? (via carbonium?ion neutralisation). The mechanisms of oxygen activation in both fungal and PPV systems are compared. In fungal hydroxylation:

Oxygenation occurs at allylic positions and double bonds (with rearrangement) Only cis products are observed A “substrate pocket” in the hydroxylase holds the substrate in a specific geometry (Fig. 1)

In the PPV system:

Substrates bind to vanadium through a double bond or an introduced hydroxyl group Greater flexibility results in racemic products for planar rings Steric factors dominate in ??pinene, ??pinene, and limonene, giving optically active products

A “hot carbonium?ion mechanism” previously proposed is discussed with reference to the behaviour of 4?methylcyclohexene and tetralin. The study of 4,4?dimethylcyclohexene suggests limitations to this hypothesis.

Section II.1 This section concerns the enzymatic and chemical deacylation of penicillin?G to 6?APA. Metal?complex design parameters include:

A hydrophobic benzyl?binding site Proximity to the target 6?amide bond Possible involvement of a metal ion in Fusarium penicillinacylase (inactivation by Zn²? chelation; activity restored by Mn²?, Cd²?, Fe²?)

Ligands chosen include dibenzoylmethane and diphenylglyoxime, whose aromatic rings enable hydrophobic association with penicillin?G.

Section II.2 This section presents:

The synthesis of metal complexes containing hydrophobic benzyl groups Their ability to associate with the benzyl group of penicillin?G (Fig. 5) The modified Kavangah assay used for measuring 6?APA The test organism used: Bacillus subtilis strain 8236

Section II.3 This section deals with spectral methods used to determine thermodynamic parameters of catalyst–substrate association. From UV absorption at ~265 nm:

Association constants K ? 190–250 L·mol?¹ Free energies ?G ? –3.1 to –3.5 kcal·mol?¹

Hydrolysis of penicillin?G was rapid but ceased after only 5–7% conversion to 6?APA, possibly due to catalyst inactivation by phenylacetic acid, which competes for hydrophobic binding.