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Titolo:
ESSENTIAL ROLE OF ARGININE-235 IN THE SUBSTRATE-BINDING OF LACTOBACILLUS-PLANTARUM D-LACTATE DEHYDROGENASE
Autore:
TAGUCHI H; OHTA T;
Indirizzi:
UNIV TOKYO,DEPT AGR CHEM,BUNKYO KU TOKYO 113 JAPAN
Titolo Testata:
Journal of Biochemistry
fascicolo: 5, volume: 115, anno: 1994,
pagine: 930 - 936
SICI:
0021-924X(1994)115:5<930:EROAIT>2.0.ZU;2-K
Fonte:
ISI
Lingua:
ENG
Soggetto:
ENZYME CATALYSIS; SITE; MUTAGENESIS; FRAMEWORK;
Keywords:
ACTIVE CENTER; D-LACTATE DEHYDROGENASE; LACTOBACILLUS PLANTARUM; SITE-DIRECTED MUTAGENESIS; SUBSTRATE BINDING SITE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
19
Recensione:
Indirizzi per estratti:
Citazione:
H. Taguchi e T. Ohta, "ESSENTIAL ROLE OF ARGININE-235 IN THE SUBSTRATE-BINDING OF LACTOBACILLUS-PLANTARUM D-LACTATE DEHYDROGENASE", Journal of Biochemistry, 115(5), 1994, pp. 930-936

Abstract

Substitutions of the conserved Arg-235 with Lys and Gin induced drastic decreases in the catalytic efficiency of Lactobacillus plantarum D-lactate dehydrogenase (D-LDH). Both the mutant enzymes showed a markedresistance to 2,3-butanedione, by which the wild-type enzyme is rapidly inactivated unless NADH and oxamate are present. The pK(a) of the catalytic His was markedly shifted to the alkaline side by the Arg-to-Gln substitution, while it was not significantly shifted by the Arg to Lys substitution. The Arg-to-Lys replacement, by which the catalytic efficiency mas less damaged, also induced decreases in k(cat)/K-m for alternative substrates, such as 2-ketobutyrate, by approximately 10,000-fold, virtually the same level as in the case of pyruvate. Although both the mild-type and mutant enzymes exhibited lower k(cat)/K-m for the alternative substrates than that for pyruvate, in the case of the mutant enzyme, the decrease in k(cat)/K-m for the alternative substrateswas mostly due to a decrease in k(cat), while it was caused mainly byan increase in K-m in the wild-type enzyme, suggesting that the mutant enzyme tends to form a nonproductive enzyme-substrate complex, in particular with more unfavorable substrates. The pH-dependence of the kinetic constants also indicated that there is a nonproductive binding that does not require the protonated or deprotonated form of the catalytic His residue. These results strongly suggest that Arg-235 plays an essential role in the tight and correct binding of substrate to the binding site of D-LDH, as in the case of Arg-171 in L-LDH.

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Documento generato il 26/11/20 alle ore 19:55:13