Studies on determination of active site amino acid residues in glyoxylate synthetase from potato tuber chloroplasts

A homogeneous preparation of glyoxylate synthetase from greening potato tub ers was used to study the functional role of disulphide groups, lysine and tryptophan residues in enzyme catalysis. The formation of a thioisoindole d erivative was demonstrated by spectral analysis of the reduced and o-phthal aldehyde-treated enzymes. o-Phthalaldehyde modification resulted in about a 25% loss of tryptophan emission at 336 nm and the appearance of a 410-nm e mission peak characteristic of a thioisoindole. Ferrous iron was capable of generating thiol groups and addition of substrate resulted in a faster dis appearance of these thiols. The optimal time for maximum glyoxylate synthes is by glyoxylate synthetase paralleled the disappearance of these thiols. I nvolvement of lysine and tryptophan residues in the enzyme reaction was dem onstrated by the inhibition of activity by pyridoxal 5'-phosphate and dimet hyl(2-hydroxy 5-nitrobenzyl) sulphonium bromide (DMHNB), respectively. Pyri doxal phosphate strongly and reversibly inhibited glyoxylate synthetase, an d substrate and metal ion provided significant protection against inhibitio n. The results suggest that the lysine residue may be at or near the active binding site. The lysyl residue formed a Schiff base with pyridoxal phosph ate which was stabilised by NaBH4. Glyoxylate synthetase was also irreversi bly inactivated by a tryptophan selective reagent, DMHNB, while substrate p rovided substantial protection against inactivation. Kinetic analysis and c orrelation of the spectral data at 410 nm indicated that complete inactivat ion by DMHNB resulted from the modification of 5 tryptophan residues/subuni t, of which one was essential for activity. The available evidence suggests a possible concerted action of enzyme disulphides, ferrous iron, lysine an d aromatic amino acid residues in the synthesis of glyoxylate by this enzym e. (C) Elsevier, Paris.