Molecular asymmetry in spinach leaf glyceraldehyde 3-phosphate dehydrogenase: interaction of the enzyme with its coenzyme & SH-reagents
Malhotra, O.P.; Agrawal, K.
Indian Journal of Biochemistry and Biophysics 19(5): 314-319
1982
ISSN/ISBN: 0301-1208 PMID: 7184847 Document Number: 196437
Interaction of spinach leaf glyceraldehyde 3-phosphate dehydrogenase (GPDH) with NAD+ results in the appearance of a new weak UV absorption band (.epsilon.max320 nm, 925 cm-1 l mol-1 bound NAD+). The binding isotherm of NAD+ to GPDH monitored at 320 nm suggests that the tetrameric enzyme molecule has 2 pairs of NAD+ binding sites (total 4 sites for MW 155,000) of unequal affinity (dissociation constants 25 .mu.M and 180 .mu.M, respectively). Spinach leaf GPDH has 4 reactive SH groups/mol tetrameric enzyme. Treatment of the enzyme (0.05 .mu.M) with excess iodoacetate (10 .mu.M and 20 .mu.M) leads to an exponential decay of enzyme activity, whereas inactivation with excess N-ethyl maleimide (10 .mu.M and 20 .mu.M) exhibits biphasic kinetics, wherein a fast and a slow phase each accounts for half of the initial enzyme activity. NAD+ slows down the time-dependent activity loss, without affecting the biphasic kinetics. With pCMB [p-chloromercuribenzoate] (0.54 .mu.M with 0.006 .mu.M enzyme), half of the activity is destroyed rapidly and the remaining half is stable for prolonged periods. Molecular asymmetry apparent in the binding isotherm of NAD+, and inactivation with N-ethyl maleimide and pCMB were attributed to the unique quaternary structure of the enzyme.