Identification of a critical lysine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cell - Comparison with the rabbit muscle enzyme

The involvement of the lysine residue present at the active site of Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (Gra 3PDH) was investigated by using the lysine specific reagents trinitrobenzen esulfonic acid (TNBS) and pyridoxal phosphate (PP). Both TNBS and PP inacti vated EAC cell Gra3PDH with pseudo-first-order kinetics with the rate depen dent on modifier concentration. Kinetic analysis, including a Tsou plot, in dicated that both TNBS and PP apparently react with one lysine residue per enzyme molecule. Two of the substrates, D-glyceraldehyde-3-phosphate and NA D, and also NADH, the product and competitive inhibitor, almost completely protected the enzyme from inactivation by TNBS. A comparative study of Gra3 PDH of EAC cell and rabbit muscle indicates that the nature of active site of the enzyme is significantly different in these two cells. A double inhib ition study using 5,5'-dithiobis(2-nitrobenzoic acid) and TNBS and subseque nt reactivation of only the rabbit muscle enzyme by dithiothreitol suggeste d that a cysteine residue of this enzyme possibly reacts with TNBS. These s tudies on the other hand, confirm that an essential lysine residue is invol ved in the catalytic activity of the EAC cell enzyme. This difference in th e nature of the active site of EAC cell Gra3PDH that may be related to the high glycolysis of malignant cells has been discussed.