alpha-chymotrypsin and tryptophan-methyl-ester

We studied how tryptophan methyl ester and related compounds inhibit binding of estrone to rat alpha-fetoprotein and find that: (a) like chymotrypsin, alpha-fetoprotein binds tryptophan esters with higher affinity than tryptophan or its amides; (b) the affinity of alpha-fetoprotein for tryptophan methyl ester is 3.7 . 10(-4) M, which is close to the affinity of chymotrypsin (10(-4) M); (c) alpha-fetoprotein binding of tryptophan methyl ester is stereoselective and pH dependent. All of these observations suggest that there is a specific interaction between alpha-fetoprotein and the chymotrypsin substrate, tryptophan methyl ester, and that rat alpha-fetoprotein contains a site with some structural similarities to the catalytic site in chymotrypsin. Since we also find that tryptophan methyl ester is a competitive inhibitor of estrone binding to alpha-fetoprotein, it is possible that the protease substrate binding site on alpha-fetoprotein is spatially close to the estrone binding site.

Topics: alpha-Fetoproteins; Animals; Chymotrypsin; Estrone; Kinetics; Protein Binding; Rats; Tryptophan

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.

Topics: Alanine; Amino Acid Oxidoreductases; Amino Acids; Animals; Biochemical Phenomena; Biochemistry; Catalysis; Chemical Phenomena; Chemistry, Physical; Chymotrypsin; Glycine; Hydroquinones; Kinetics; L-Amino Acid Oxidase; Phenylalanine; Research; Snakes; Tryptophan; Venoms