Spectrophotometric identification of an active site-specific acyl glyceraldehyde 3-phosphate dehydrogenase. The regulation of its kinetic and equilibrium properties by coenzyme.

Glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle reacts rapidly with /3-(2-furyl)acryloyl phosphate (FA-phosphate) in aqueous solutions at neutral pH to yield an active site-specific, spectrophotometrically identitlable /3-(2-furyl)acryloyl enzyme (FA-enzyme). This acyl-enzyme reacts with arsenate, phosphate, and NADH to give p-(2furyl)acrylic acid, FA-phosphate, and P-(2-furyl)acrolein, respectively. The ultraviolet spectrum of native FA-enzyme includes, in addition to the protein absorption bands, a strong absorption band with X,,, at 344 rnp. On denaturation this absorption maximum shifts to the maximum observed for a corresponding furylacryloyl thiol ester (337 mp). The formation of acyl-enzyme in excess of FA-phosphate follows a single exponential (6rst order) time dependence. The rate of acylation is a function of [PA-phosphate] and [NAD+]. k acylation increases linearly with [NAD+] in the concentration range in which 0.5 to 2.0 moles of NAD+ are bound per mole of enzyme (mol wt 140,000), and extrapolates to zero, at zero NAD+ concentration. In the presence of a large excess of acyl phosphate, only 2 eq of acyl group can be introduced per mole of glyceraldehyde-3-P dehydrogenase, although this enzyme is known to be made up of four subunits of identical primary structure. At very high [FA-phosphate] and low ]NAD+], the stoichiometry can be raised to as high as 3.5 eq of acyl per mole of enzyme; the tirst 2 acyl equivalents are introduced about 20 times more rapidly than subsequent acyl groups. Stoichiometric considerations and the influence of [NAD+] on kacylation strongly suggest that the four subunits (CZ) of the glyceraldehyde-3-P dehydrogenase molecule are arranged as two pairs (interacting pairs) (C&J rather than as a symmetrical tetramer ((Ye). The atiity of the enzyme for NAD+ decreases on acylation. Increasing [NAD+] shifts the equilibrium, FA-phosphate + E = FA-enzyme + Pi, toward