Nonessentiality of Histidine 291 of Rhodospirillum rubrum

Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential activesite residue (Igarashi, Y., McFadden, B. A., and ElGul, T. (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pKa of His-298 was observed to be -6.8, and it was uggested that this histidine might be the essential base that initiates catalysis (Paech, C. (1985) Biochemistry 24, 31943199). To explore further the possible function of His298, we have used site-directed mutagenesis to replace the corresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is -40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a kcat of 1.5 s-’ and a k,, JK,,, of 1.7lo4 M” s-l in contrast to values of 3.6 s” and 6-10‘ M” s-l for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.