Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

Publisher Website
Google Scholar
Abstract
The binding of NAD+ and NADH to [sturgeon] 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12] was studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25%) and the induction of a difference spectrum in the UV absorbance region of the enzyme. Both spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme-NAD+ complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to 4 NAD+ molecules are bound to the enzyme tetramer. No anticooperativity was detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of 4 binding sites with a Kd of 25 .mu.M at 10.degree. C, pH 7.0. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. A large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme-NADH complex, since it disappears upon arsenolysis of the acylenzyme. The binding affinity of NADH to the acylated enzyme was estimated by simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD+ to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD+, NADH appears to be strongly bound to the acylated enzyme. The Kd of the acylenzyme-NADH complex was estimated as 2.0 .mu.M at 25.degree. C. A large quenching of the NADH fluorescence (about 83%) is observed. A reaction mechanism in which significant formation and dissociation of NAD+-acylenzyme and NADH-acylenzyme complexes occur is suggested. Under physiological conditions the activity of the enzyme is regulated by the ratio of oxidized and reduced coenzymes. Reasons for the lack of anti-cooperativity in coenzyme binding to the acylated form of the enzyme are discussed.