Effects of salts on thermolysin: activation of hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester, and a unique change in the absorption spectrum of thermolysin.
It has been reported that neutral salts such as NaCl activate the thermolysin-catalyzed hydrolysis of substrates containing glycine at the P1 position (carboxylic side of the cleavage bond) [Holmquist, B. & Vallee, B.L. (1976) Biochemistry 15, 101-107]. In this paper, we demonstrate that high concentrations (1-4 M) of neutral salts greatly enhance the thermolysin activity in both hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZAPM), a precursor of a peptide sweetener, aspartame, in which the L-aspartyl residue is the P1 residue. The enzyme activity is enhanced with an increase in salt concentration in a pseudo-exponential fashion. The degree of activation by salts was in the order LiCl > NaCl > KCl. The rate of ZAPM hydrolysis in the presence of 3.8 M NaCl was 6-7 times higher than that in its absence, and 50 times or more activation is expected in saturated NaCl solution. The activation is brought about solely through an increase in the catalytic constant (kcat), and the Michaelis constant (Km) is not affected at all by the presence of NaCl. On mixing thermolysin with NaCl, a unique absorption difference spectrum suggesting a conformational change of the enzyme was observed. The intensity increased in a pseudo-exponential fashion with increase of NaCl concentration up to 3 M, and this dependence is similar to that of the enzyme activity.
Inouye
K
K
Biotechnology Research Laboratories, Tosoh Corporation, Kanagawa.
D001407:Bacillus / Q000201:enzymology; D004151:Dipeptides / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D012492:Salts / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013820:Thermolysin / Q000737:chemistry / Q000378:metabolism*
D004151:Dipeptides; D012492:Salts; C051210:N-benzyloxycarbonyl-aspartylphenylalanine methyl ester; D013820:Thermolysin