OPHIDIAN l-AMINO ACID OXIDASE. THE NATURE OF THE ENZYME-SUBSTRATE COMPLEXES

Abstract

1. To investigate the kinetics of ophidian l-amino acid oxidase, V and K(m) were determined for phenylalanines that were substituted in every ring position with groups of various size and reactivity, and for a few ring-substituted tryptophans and histidines. The venom of one representative from each of three major classes of poisonous snakes, Naja melanoleuca, Vipera russelli and Crotalus adamanteus, served as a source of the ophidian l-amino acid oxidase. Both crude and crystalline enzyme from the venom of C. adamanteus were tested. 2. The introduction of a benzene ring into glycine and alanine caused some increase of V and a very marked depression of K(m). 3. With the exception of fluorine, residues in the ortho position of phenylalanine led to a decrease of V. The rates induced by various substitutions follow the pattern: meta >/= para >/= ortho. Within the halogen series, the effects become more pronounced with increasing atomic number. 4. Ring substitution in heterocyclic amino acids also affected the V values markedly. For methyl-substituted tryptophans the pattern was: 5-methyl >/= 6-methyl >/= 4-methyl. In a few instances ring substitution accounts for a considerable elevation of V, as shown for beta-quinol-4-ylalanine and its 6-methoxy derivative. 5. The kinetic constants appear to be unaffected by relatively high concentrations of the corresponding d-amino acids. 6. A general principle that permits a uniform interpretation of a vast body of information is suggested. It is based on the assumption that most substrates form not only eutopic but also dystopic complexes with the enzyme. The latter, in contrast with the former, do not permit the formation of reaction products. K values for eutopic and dystopic complexes are computed. Similar concepts have been presented to elucidate the action of alpha-chymotrypsin (Hein & Niemann, 1962) and of monoamine oxidase.

Interaction of ophidian L‐amino acid oxidase with its substrates and inhibitors: Role of molecular geometry and electron distribution. Communication 6 on ophidian L‐amino acid oxidases

“…The existence of hydrophobic forces in the formation of the enzyme-substrate complex has been previously postulated [4]. This conclusion is reinforced by the results presented here which reveal linear correlations between Hansch's hydrophobic constant z and the Ki-values obtained for four sets of competitive inhibitors.…”

“…The origin and synthesis of the substrates has previously been reported [4]. The substituted phenylacctic acids were obtained from K & K I,aboratories, Inc., Plainview, N. Y .…”

“…The apparatus was equipped with a membrane-covercd electrode of the Clark type [17] which was connected to a Bausch & Lolnb VOM 5 recorder operating at 100 mv. Thc manometric proccdurc was described previously [4].…”

“…For log K," -log K y = a n f b (4) the para-substituted compounds b N 0, while for the meta-derivatives, b < 0.…”

“…7 , first described by Zeller & Maritz [S-lo], seemed to be a suitable system for providing additional information about the existence of multiple enzyme-substrate complexes. Previously, several para-substituted phenylalanines wzre shown to be much more slowly degraded by crude and purified L-AAO than the corresponding meta-isomers [4]. From these and other observations it was tentatively concluded that large para-residues force the substrate molecule into non-productive complexes.…”