Keto-enamine Form Research Articles

PH Jump Studies of Glutamate Decarboxylase: EVIDENCE FOR A pH-DEPENDENT CONFORMATION CHANGE

The catalytically active form of bacterial glutamate decarboxylase has an absorption maximum at 420 nm due to the presence of the enzyme—pyridoxal 5′-phosphate Schiff base. Above pH 5.5 the absorption maximum is at 340 nm, and the enzyme is inactive. If the pH of a solution of the enzyme is rapidly changed from 6.5 to 4.0 in a stopped flow apparatus, the change in absorption spectrum occurs over a period of several seconds. The change is general acid catalyzed and occurs in two steps. The first is a rapid conformation change of the enzyme induced by the addition of 3 protons to the enzyme. The second is general acid catalyzed formation of the catalytically active form of the enzyme. If the pH of a solution of the enzyme is rapidly increased from 4.0 to 7.0, the rate of the change in absorption spectrum can also be measured. The change is not buffer catalyzed, but its rate increases rapidly with increasing pH. The mechanism of this change involves an initial rate-determining conformation change of the enzyme induced by dissociation of 3 protons, followed by rapid formation of the equilibrium high pH form of the enzyme. The rate of the conformation change can also be measured on enzyme which has been reduced by sodium cyanoborohydride. From the kinetics of the pH jump reactions and other factors we conclude that the high pH form and the low pH form of glutamate decarboxylase differ by a protein conformation change and by a change in the covalent structure of the bound coenzyme. At low pH the coenzyme is bound in the usual ketoenamine form. At high pH a sulfhydryl group of the enzyme adds to the aldehyde carbon forming an aldamine. Many anions affect the properties of glutamate decarboxylase. Chloride ion increases the rate of transformation of the high pH form of the enzyme into the low pH form and decreases the rate of transformation of the low pH form into the high pH form. These effects occur because anions are able to bind to the low pH conformation of the enzyme but not to the high pH conformation.

Factors Contributing to the Absorption and Fluorescence Characteristics of Pyridoxal Phosphate in Glycogen Phosphorylase b

AbstractThe Schiff base formed between salicylaldehyde and n‐hexylamine has absorption bands peaked at 255 and 317 nm in non‐polar solvents. In a polar solvent (ethanol) an additional band appears at 405 nm and a shoulder is resolved at 275 nm. The fluorescence band has a peak at 535 nm in non‐polar solvents (with the exception of CCl4) which is blue shifted to 505 nm in ethanol. These spectral data are similar to those of Schiff bases formed between pyridoxal phosphate and aliphatic amines. On the other hand, the spectral properties of the Schiff base of o‐methoxybenzaldehyde with n‐hexylamine are very different. It may thus be concluded that the nitrogen atom of the aromatic ring does not have to be invoked in explaining the spectral properties of pyridoxal phosphate Schiff bases and of pyridoxal phosphate in glycogen phosphorylase. The hydroxyl group seems however to play an important role in determining the spectral properties of Schiff bases of o‐hydroxyaldehydes. The optical rotatory power of the pyridoxal phosphate in phosphorylase is of comparable magnitude for the chromophore in the ground state or the electronically excited state, indicating similarity of mode of binding of the chromophore to the protein in the two electronic states. Measurements of circular dichroism indicate that the “deformer” imidazole citrate affects the interaction of the pyridoxal phosphate with the protein when the cofactor Schiff base is in the ketoenamine form but not when in the enolimine form.