Kinetic Studies on the Action of Carboxypeptidase A on Bovine Insulin and Related Model Peptides^*

Abstract

Several model peptides which have carboxylterminal amino acids related to those found in bovine insulin were synthesized and tested as substrates for the action of carboxypeptidase A over the pH range 6.0-9.0. The normalized initial velocities (v/[EI,) for the action of the enzyme on the carbobenzoxyglycyl derivatives of L-alanine, L-glutamine, and L-asparagine were directly proportional to the substrate concentrat ion over the substrate range 0.01-0.15 M in the pH ritnge 6.5-9.0. The second-order rate constants ( k ) for these substrates exhibited maximum values at pH 7.0-7.5. Carbobenzoxyglycyl derivatives of L-glutamic acid and Laspartic acid were much poorer substrates than the corresponding amides. The initial velocities for the action of carboxypeptidase A on these substrates had maximum values at pH 6.0 (or below) and fell off rapidly at pH values above 7.0. The difference in the pH optimum between the neutral and acidic: amino acid residues explains in part the differences previously encountered in the pH profile for the carboxypeptidasecatalyzed hydrolysis of asparagine or aspartic acid from insulin and desamido-insulin, respectively. In studies on insulin the rate of release of carboxyl-terminal alanine (from the B chain) and of asparagine (from the A chain) was markedly increased as the concentration of the insulin was lowered from 10 mg/rril to 1 mg/ml C arboxypeptidase A has been used extensively as a tool for elucidating the primary structurl: of proteins, and much valuable information about its mode of attack on proteins has been accumulated i: Davie et at., 1959;Neurath, 1960). The present investigation stems from earlier work in this laboratory in which carboxypeptidase A was used to determine the difference in the primary structure between insulin and desamido-insulin (Slobin and Carpenter, 1963a,b). We were attracted to a study of the kinetics of the insulin-carboxypeptidase A system by several features which made it appear suitable Congress of Biochemistry (1964). Abstracted from a thesis submitted by Lawrence I. Slobin in partial fulfillmerit of the requirements of the Ph.D. degree at the University of California, Berkelev. 1964. in the pH range 7.5-9.0. The rate of release of both amino acids increased in going from pH 7.5 to 9.0 at all concentrations of insulin. Furthermore, increasing the salt concentration brought about a decrease in the rate of release of carboxyl-terminal amino acids from insulin. These results on insulin are in contrast to those predicted from studies of the action of carboxypeptidase on model peptides. However, there is a good correlation bet ween conditions which bring about depolymerization of insulin (as judged by ultracentrifugal studies) and those which increase the rate of attack of carboxypeptidase on insulin. This correlation suggests that the action of carboxypeptidase on insulin is largely dependent on the degree of polymerization of the insulin.Rotatory dispersion studies have been made of the products of digestion : desalanine-insulin and desalanine-des...

The case for an ?abnormal? insulin in diabetes mellitus

“…Disruptive changes in the secondary structure of the molecule affects the ability of the molecule to aggregate [39]. The recent evidence favouring the presence of an insulin precursor in isolated islets of Langerhans might be a very attractive explanation for the "abnormal insulin concept".…”

Fluorine nuclear magnetic resonance studies of trifluoroacetyl-insulin derivatives. Effect of pH on conformation and aggregation

“…The l9F spectrum of the glycineA"1-phenylalanine®"1trifluoroacetyl derivative of this compound at pH 6.8 supported this proposal, affording a well-resolved two-peak spectrum (Table II, Figure 3f). Several studies have suggested that insulin disaggregates at pH values above 8 (Fredericq, 1956;Slobin and Carpenter, 1966). Accordingly, glycineA"'-phenyl-alanine®' '-trifluoroacetyl-insulin and glycineA"1 -phenylala-nine®" 1-lysine®"29-trifluoroacetyl-insulin were studied at pH 8.7.…”

“…Various studies have been undertaken to explore this complex behavior, utilizing sedimentation velocity and equilibrium measurements (Jeffrey and Coates, 1966a,b;Pekar and Frank, 1972; Slobin and Carpenter, 1966;Fredericq, 1956), ultraviolet spectroscopy (Rupley et al, 1967;Frank et al, 1972;Lord et al, 1973), and optical rotatory dispersion and circular dichroism (Carpenter, 1966;Levy and Carpenter, 1970;Morris et al, 1968;Mercóla et al, 1967Mercóla et al, , 1972Ettinger and Timasheff, 1971). The availability of the high-resolution X-ray crystallographic data for zinc insulin (Blundell et al, 1972;Adams et al, 1969) further enables the analysis of the results obtained from these solution studies.…”

Wege zu neuen Enzymen: Protein Engineering und Katalytische Antikörper

“…Bereits wenige Jahre spater wurde diese Idee von mehreren Arbeitsgruppen unabhangig voneinander, aber in allen Fallen mit nur magigern Erfolg getestet [67]. Der Miflerfolg beruhte wahrscheinlich darauf, daR die durch Antikorper verursachte Reaktionsbeschleunigung sehr klein ist.…”