Ribulose Bisphosphate Carboxylase/Oxygenase: Active-Site Characterization and Function of Critical Residues

摘要

An emphasis has been placed on affinity labels to probe the active site of ribulose-P sub 2 carboxylase because of their inherently high degree of specificity, which simplifies correlation of inactivation with modification. Collective data provided by several different affinity labels have revealed the presence of two distinct lysyl residues at the active site. The absolute species invariance of both lysines and the sequence homologies of adjacent regions among the carboxylases from evolutionarily diverse organisms suggest that the two active-site lysines are directly or indirectly required for proper enzyme functions. These homologies are especially striking in view of the total sequence homology of only 31%. Another affinity label suggests that the active-site domain also includes a region of the polypeptide near the NH sub 2 -terminus. This reagent labels both His-44 and Cys-58 of the R. rubrum enzyme in a mutually exclusive fashion. Neither residue can be essential since they are not species invariant. However, the high degree of homology within this region indicates a prominent role for one or more of the conserved residues. The recently reported 3D structure of the dimeric R. rubrum enzyme places the NH sub 2 -terminal segment of one subunit near the active-site Lys-166 of the adjacent subunit. Thus, the possibility arises that the active site is positioned at a subunit interface and is comprised of segments of polypeptide from both subunits. Consistent with this possibility is the observation that 4,4'-difluoro-3,3'-dinitrophenyl sulfone, a bifunctional reagent with a span of 9 A, forms an intersubunit crosslink between Lys-166 and Cys-58. 26 refs., 2 figs. (ERA citation 12:027441)