Histidyls in lobster arginine kinase

Abstract

The role of histidyls in lobster arginine kinase (EC 2.7.3.3) has been studied by 1H-NMR spectroscopy of the enzyme and its complexes with substrates or their analogues and 31P-NMR spectroscopy of complexes with ADP. Five histidyls were detected by 1H-NMR in native enzyme (His 1 to His 5). Three of them appeared possibly to be implicated in catalysis: His 3, whose pH/titration was affected by arginine binding, and His 1 and 4, shown from paramagnetic relaxation by Mn2+ to be close (less than or equal to 1.2 and less than or equal to 1.27 nm respectively) to the metal cofactor. His 4 was broadened beyond detection in the presence of any adenine nucleotide. In the enzyme reversibly inactivated by histidine ethoxyformylation, the modified histidyl was His 1. In the transition state analogue complex (in which NO3- mimics the transferred phosphoryl), Hill plots of histidyl pH/titration curves showed that His 1 and His 3 were both interacting with the same set of three titratable groups and hence spatially close. 31P-NMR demonstrated that ADP binding in this complex was unaffected by the chemical modification of His 1. It is concluded that His-ethoxyformyl-enzyme is inactive because ethoxyformyl-His 1 is unable to titrate. This is consistent with His 1 acting as the acid-base catalyst. However our results, which do not indicate any catalytic role of His 3, exclude any H-bonding of His 1 on either substrate. Involvement is needed of at least one other titratable residue for the proton evolved in the catalysis to exchange directly with the guanidino substrate.