Glyceraldehyde-3-phosphate dehydrogenase of Scenedesmus obliquus. a kinetic analysis of the effects of nucleotide and dithiothreitol on the production of NADPH-dependent activity

Woodrow, S.; O'Brien, M.J.; Easterby, J.S.; Powls, R.

European Journal of Biochemistry 98(2): 425-430

1979


ISSN/ISBN: 0014-2956
PMID: 39754
DOI: 10.1111/j.1432-1033.1979.tb13202.x
Accession: 068518720

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Abstract
Incubation of the hexadecameric glyceraldehyde-3-phosphate dehydrogenase of S. obliquus with dithiothreitol causes a transient increase in NADPH-dependent activity. When NADPH is also incorporated in the incubation buffer, the induced NADPH-dependent activity is stabilized. In the presence of NADPH, the rate of induction of NADPH-dependent activity is directly proportional to dithiothreitol concentration. This is consistent with either a dithiothreitol-mediated reduction of disulphide bonds or dithiothreitol-promoted disulphide exchange in the enzyme without prior formation of a non-covalent complex between enzyme and reductant. A kinetic analysis of the transient induction of the NADPH-dependent activity by dithiothreitol alone shows that the activation is thiol-dependent with a 2nd-order rate coefficient of 4.3/M per min. In contrast, the ensuing inactivation is spontaneous and does not involve thiol. It proceeds with a 1st-order rate coefficient of 0.032/min, corresponding to a half-time of 22 min. Dissociation of the hexadecameric enzyme to the tetramer is promoted by dithiothreitol whereas further dissociation to an inactive dimer results purely from the inherent instability of the tetramer in the absence of NADPH. The rate of induction of NADPH-dependent activity by dithiothreitol increased hyperbolically with respect to NADPH concentration, implying that NADPH has more than a stabilizing role in the activation process. These observations are discussed in relation to the photoreductive process thought to occur in vivo.