Kinetic evidence for a reversible isomerization of pig muscle glyceraldehyde-3-phosphate dehydrogenase in its crystallization medium

M. Vas, Rodolfo Berni, József Batke, Tamás Keleti, Gian Luigi Rossi

Research output: Contribution to journalArticle

Abstract

Ammonium sulfate, a typical component of crystallization media of proteins, stabilizes an inactive conformation of pig muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, in the presence of ammonium sulfate the reconstitution of the catalytically active holoenzyme from the apoenzyme and NAD is not instantaneous, as in the case of enzymes from Bacillus stearothermophilus and the Mediterranean lobster Palinurus vulgaris. With pig muscle enzyme, at pH 6.0, the time course of formation of the characteristic Racker band can be monitored by a rapid mixing stopped flow technique. Activation follows a single exponential curve with a rate constant independent of the concentration of both NAD and protein and, therefore, appears to be limited by a slow protein isomerization (k = 7 ± 2 s-1). Accordingly, when the apoenzyme is simultaneously exposed to NAD and either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate, the ensuing reactions (the redox and the acylation steps, respectively) are kinetically limited by the same protein isomerization. At pH 7.0 and 8.0, however, two among the four active sites react with NAD at an unmeasurably high rate, while the other two sites behave as they do at pH 6.O. When the pig muscle apoenzyme is preincubated and allowed to react with either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate before the rapid mixing with NAD, both the redox reaction and the NAD-dependent activation of apo-acyl-enzyme toward arsenolysis become unmeasurably fast. Similarly, when the sulfate in the medium is replaced by ions such as phosphate and citrate, the reconstitution of the active holoenzyme is practically instantaneous. Thus, the slow protein isomerization observed in the presence of sulfate and abolished by competing substrates and anions is diagnostic of a structural state of the pig muscle apoenzyme, which is induced by sulfate ions bound within the enzyme active site.

Original languageEnglish
Pages (from-to)121-129
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume263
Issue number1
DOIs
Publication statusPublished - May 15 1988

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Glyceraldehyde-3-Phosphate Dehydrogenases
Isomerization
Crystallization
NAD
Apoenzymes
Muscle
Swine
Muscles
Kinetics
Glyceraldehyde 3-Phosphate
Sulfates
Holoenzymes
Ammonium Sulfate
Enzymes
Proteins
Oxidation-Reduction
Catalytic Domain
Chemical activation
Ions
Geobacillus stearothermophilus

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Kinetic evidence for a reversible isomerization of pig muscle glyceraldehyde-3-phosphate dehydrogenase in its crystallization medium. / Vas, M.; Berni, Rodolfo; Batke, József; Keleti, Tamás; Rossi, Gian Luigi.

In: Archives of Biochemistry and Biophysics, Vol. 263, No. 1, 15.05.1988, p. 121-129.

Research output: Contribution to journalArticle

Vas, M. ; Berni, Rodolfo ; Batke, József ; Keleti, Tamás ; Rossi, Gian Luigi. / Kinetic evidence for a reversible isomerization of pig muscle glyceraldehyde-3-phosphate dehydrogenase in its crystallization medium. In: Archives of Biochemistry and Biophysics. 1988 ; Vol. 263, No. 1. pp. 121-129.
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