Inhibition by Pyridoxal‐Phosphate of Glyceraldehyde‐3‐Phosphate Dehydrogenase

Experiments have been carried out in order to investigate the effect of pyridoxal‐5′‐phosphate on glyceraldehyde‐3‐phosphate dehydrogenase. It was found that the oxidation of glyceraldehyde‐3‐phosphate is inhibited by low concentrations (0.1 mM) of pyridoxal‐5′‐phosphate preincubated with the enzyme. The inhibition increases with higher pyridoxal‐5′‐phosphate concentration, and is removed by the addition of substrate, lysine or valine. The reversibility of the inhibition was substantiated by dilution techniques. Pyridoxal was not found to substitute for pyridoxal‐5′‐phosphate in producing the inhibition. The difference spectra of the pyridoxal‐5′‐phosphate‐treated enzyme show the appearance of two characteristic peaks at 378 and 430 mμ, related to the Schiff bases. Among the products of hydrolysis of the pyridoxal‐5′‐phosphate‐treated enzyme reduced with borohydride, the presence of ɛ‐pyridoxyl‐lysine was detected by spectrophotometric and chromatographic analysis. It was found that the number of the pyridoxal‐5′‐phosphate‐bound NH2‐lysyl residues increases up to 10–11 moles per mole of enzyme with increasing pyridoxal‐5′‐phosphate concentration in the preincubation mixture. Comparison of the enzymic activity with the numbers of the pyridoxal‐5′‐phosphate‐bound NH2‐lysyl residues suggests that the activity decreases proportionally with the increase of the pyridoxal‐5′‐phosphate‐linked groups. All the results, which have been reported, suggest that NH2‐lysyl residues of glyceraldehyde‐3‐phosphate dehydrogenase may be an essential requirement for the oxidative activity of the enzyme.