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

Journal of Biochemistry
Kuniyo Inouye

Abstract

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 i...Continue Reading

Citations

Dec 14, 2006·Biotechnology and Bioengineering·Johanna Mansfeld, Renate Ulbrich-Hofmann
May 16, 2008·The FEBS Journal·Karolis VaitkeviciusSun N Wai
Dec 3, 2009·Physical Chemistry Chemical Physics : PCCP·Jan HeydaJirí Vondrásek
Oct 23, 2012·Journal of Biochemistry·Yuichi KawasakiKuniyo Inouye
Jul 10, 2007·Bioscience, Biotechnology, and Biochemistry·Kuniyo InouyeKiyoshi Yasukawa
Aug 17, 2011·Applied Microbiology and Biotechnology·Ji-Wei Wu, Xiu-Lan Chen
Dec 6, 2011·Applied Biochemistry and Biotechnology·Y MatsumiyaMotoki Kubo
Sep 12, 2014·Bioscience, Biotechnology, and Biochemistry·Vimbai SamukangeKuniyo Inouye
Nov 21, 2012·Angewandte Chemie·Roberto CastangiaSabine L Flitsch
Apr 20, 2005·Letters in Applied Microbiology·Y MatsumiyaM Kubo
Apr 9, 2013·Bioscience, Biotechnology, and Biochemistry·Evans MenachKuniyo Inouye

Related Concepts

N-benzyloxycarbonyl-aspartylphenylalanine methyl ester, (L-beta-Asp)-isomer
Dipeptides
Enzyme Activation
Hydrolysis
Protein Conformation
Salts
Spectrophotometry, Ultraviolet
Substrate Specificity
Thermolysin
In Vitro [Publication Type]

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