A kinetic study of the hydroxylation of monophenols catalysed by spinach leaf phenolase

McIntyre, Rosemary Jean (1973) A kinetic study of the hydroxylation of monophenols catalysed by spinach leaf phenolase. PhD thesis, University of Glasgow.

Abstract

The reaction catalyzed by spinach leaf phenolase has been shown to be complex, but little is known about the mechanism of action of this enzyme. The present investigation was undertaken in an attend to clarify the molecular interactions involved, using the techniques of steady state kinetics. Phenolase (EC: 1.3.10.1., oxygen o-diphenol oxidoreductase) was prepared in bulk by a modification of the method in use in the laboratory. A continuous spectrophotometrio assay for the determination of the reaction product, caffeic acid, was devised. Product formation could then be monitored at 340 nm in the absence of electron donor, or in the presence of ascorbic acid as reducing agent. With dimsthyltetrahydropteridlne (DMTP) or NADH as electron donor, absorbance changes were monitored at 340 and 370 nm, and 340 and 370 nm respectively. The changes in absorbance were related to caffeic acid concentration. Assay conditions were investigated, the pH of the reaction mixture and enzyme concentration being adjusted to give suitable reaction rates. The Michaelis constants for the substrates, oxygen, acid and electron donor were determined in the presence of saturating amounts of the other two substrates. The nature of the electron donor did not seem to affect the for oxygen, but did affect that of-j-coumaric acid. The determination of those constants was a necessary preliminary to a steady state kinetic analysis of the enzyme. Initial velocity studies were carried out by varying one substrate ln1he presence of fixed amounts of the other two substrates, fixed in the ratio of their Michaelis constants. The initial velocity patterns thus obtained demonstrated the existence of irreversible steps on binding of each of the three substrates, and suggested that product formation led to the ireversal of one of these steps. Experiments carried out in the presence of a fixed amount of caffeic acid suggested that a classical ping pong step occurs on the release of caffeic acid after 2-coumaric acid hydroxylation, and before electron donor has bound to the enzyme. No effect was observed on the apparently irreversible step on binding of oxygen. These results were assessed both graphically and by computer analysis in an attend to aid the interpretation of the observed patterns. On the basis of these results a model for the reaction mechanism is proposed, involving binding of oxygen followed by acid and then electron donor. Inhibitor studies with substrate analogues and alternate substrates were used to confirm the proposed order of substrate addition. Studies with the caffeic acid analogue, 3,4-dihydroxy- benzoic acid, suggested the existence of a distinct site for caffeic acid. It is proposed that this is the site occupied by caffeic acid in the role of internal electros donor to the enzyme, the o-dihydricphenol being maintained in the reduced state by the external electron donor, ascorbic acid, NADH or DMTP. This result may explain the revival of the lag observed during hydroxylation, as caffeic acid production can occur in the presence of oxygen and jgycoumaric acid. Studies with benzoic acid and 2-hydroxybenzoic acid support the proposed mechanism, although complex results are obtained owing to the structural similarity of these compounds to caffeic acid, and consequent binding to the dihydricphenol site on the enzyme. The implication of copper in the hydroxylation of monophenols provided another area for study. Copper specific reagents were used in an attempt to elucidate the role of the metal in substrate blinding. Carbon monoxide, a 0u+ specific chelator, was found to compete directly with oxygen for the enzyme. Similar results were obtained with bathocuprolne sulphonate (BGS) which has been thought to be Cu+ specific. Dlethyldlthlocarbamat (DIECA), however, competed with caffeic acid, and would therefore seem to be acting on a different form of copper. This compound has been thought to be more specific for Oa++Tr In view of the difference spectra obtained with DIECA and BCS in the presence of enzyme, it is suggested that both Cu+ and Cu++ may be implicated in the mechanism of action of phenolase. A scheme for the mechanism of the reaction of the enzyme is proposed and its validity discussed.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: P FT Vaughan
• Keywords: Plant sciences
• Date of Award: 1973
• Depositing User: Enlighten Team
• Unique ID: glathesis:1973-73814
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73814