Structure‐Function Relationship in Rabbit Muscle Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: FOUCAULT Georges , PUDLES Julio , TRAORE Fernand , LEVILLIERS Jaqueline 
Published: March 2005

Modification of the lysine residues of rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase by 2,4,6‐trinitrobenzenesulfonic acid follows a biphasic process in the apoenzyme in which two essential lysyl groups are modified per enzyme tetramer with loss of 50% of the arsenolytic, phosphorolytic, oxidative and NADH‐X activities and 15% of the esterase activity. Further decrease in the activities was much slower even though a higher number of additional residues were being modified.

The trinitrophenylated enzyme with two modified lysine residues and 50% residual activity at pH 8.0 had the same Km for NAD+, glyceraldehyde 3‐phosphate and arsenate as well as the same number of active sites as the native enzyme.

By spectrofluorimetric and spectrophotometric techniques it was shown that this modified form of the enzyme still binds four NAD+ per mole enzyme by an anticooperative process. Ultracentrifugation and thermodenaturation studies show that the chemical modification did not affect the quaternary structure or the thermostability as compared with the native enzyme.

Fluorescence studies show only a decrease in the fluorescence emission but not displacement in the wavelength of the emission maxima. However, the modified enzyme becomes more sensitive to chymotryptic proteolysis, and the pH optimum of the enzyme activity was displaced by one unit to the acid region.

When trinitrophenylation was carried out in the presence of NAD+ two lysyl groups were protected and the rate of modification was identical to that found for the slow‐reacting lysine residues of the apoenzyme. Moreover, the loss of the enzyme activities follows a linear pattern. Trinitrophenylation of the apoenzyme in presence of glyceraldehyde 3‐phosphate or Pi does not decrease the rate of modification of the lysine residues but the enzyme activity is protected and the inactivation process is also linear.

However, by prior modification of the active site sulfhydryl groups of the apoenzyme with 1‐fluoro‐2,4‐dinitrobenzene followed by trinitrophenylation of the lysine residues, no change was observed either in the rate of modification of the lysine residues or in the inactivation rate profile as compared with the native enzyme. In both cases a 50% loss of the initial activity after modification of two lysine residues was observed. This result indicates that the ligand‐induced conformational change in the enzyme active sites does not affect the reactivity of the two lysine residues per enzyme tetramer.

From these different experimental data it is concluded that two reactive lysine residues per tetramer of rabbit muscle apo‐glyceraldehyde‐3‐phosphate dehydrogenase are essential for the different enzyme activities. They do not seem to be directly involved either in the catalytic or the substrate binding site, however, they must play an important role in the stabilisation and control of the enzyme active site.

In relationship with the reactivity of the two lysine residues, our results seem to be explained by a pre‐existing asymmetry in the structure of the rabbit muscle enzyme, rather than by an induced fit mechanism.