The active site of Rubisco

Previous affinity-labelling studies and comparative sequence analyses have identified two different lysine residues at the active site of ribulose bisphosphate carboxylaseoxygenase and have suggested that they are essential to function. The essential lysine residues occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependences of inactivations of the two enzymes by trinitrobenzene sulphonate, Lys 166 ( R. rubrum enzyme) exhibits a p K a of 7.9 and Lys 334 (spinach enzyme) exhibits a p K a of 9.0. These low p K a values, as well as the enhanced nucleophilicities of the lysine residues, argue that both are important to catalysis rather than to substrate binding. Lys 166 may correspond to the essential base that initiates catalysis and that displays a p K a of 7.5 in the pH-curve for V max / K m . Cross-linking experiments with 4,4'- diisothiocyano-2,2/-disulphonate stilbene demonstrate that the two active-site lysine residues are within 12 Å of each other (1 Å = 10 -10 m).

Identification of the apparently essential lysine residues in phospholipase C (Bacillus cereus)

Phospholipase C (Bacillus cereus) contains two apparently essential and very reactive lysine residues that may be labelled selectively by pyridoxal 5′-phosphate [Aurebekk & Little (1977) Biochem, J. 161, 159–165]. One of these lysine residues was found in the 25-amino acid N-terminal fragment liberated by CNBr digestion of the pyridoxal-labelled enzyme and identified as lysine-6. Two of the labelled peptides isolated from the chymotryptic digest of pyridoxal-labelled enzyme contained proline, suggesting that the other labelled lysine residue is situated in the same region of the primary structure as the single proline residue of the enzyme.