MECHANISTIC INSIGHTS PROVIDED BY DELETION OF A FLEXIBLE LOOP AT THE ACTIVE-SITE OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE

To evaluate the functions of flexible loop 6 at the active site of Rho dospirillum rubrum D-ribulose-1,5-bisphosphate carboxylase/oxygenase, the loop was truncated by cassette mutagenesis, whereby seven residues of the twelve-residue loop were excised and replaced by two glycyl re sidues. The purified loop-deletion mutant was totally devoid of carbox ylase activity, but retained substantial catalytic competency in the e nolization of ribulose bisphosphate (the initial Step in the overall c arboxylase pathway) and in normal processing of the six-carbon carboxy lated intermediate (the terminal steps in the overall carboxylase path -way). Hence, catalytic impairment resides predominantly at the stage of carboxylation of the initial enediol(ate), a conclusion compatible with mechanistic deductions derived from crystallographic analyses. A critical role of loop 6 in the stabilization of the transition state f or carboxylation is reinforced by the findings that the loop-deletion mutant displays preferentially compromised affinity for an analogue of the carboxylated intermediate relative to ribulose bisphosphate and t hat the mutant converts the substrate to a dicarbonyl compound as a co nsequence of beta-elimination of phosphate from the initial enediol(at e).