A STUDY OF CONSERVED IN-LOOP AND OUT-OF-LOOP GLYCINE RESIDUES IN THE LARGE SUBUNIT OF RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE BY DIRECTED MUTAGENESIS/

The replacement of all 22 completely conserved glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase from Ana cystis nidulans by directed mutagenesis is described. In each beta/alp ha barrel of the large subunit there are 12 completely conserved glyci nes in six of eight loops at the C-termini of eight beta-strands and f our in loops at N-terminal ends of the beta-strands. Two completely co nserved glycines are also in each pla barrel backbone and four more ar e in a large N-terminal portion preceding the barrel in a given L subu nit. Substitution of glycines in loops that are C-terminal to beta-str ands by proline was more deleterious to carboxylase activity than that by alanine supporting the postulates that these loops contribute to c atalysis and substrate binding and that in some cases the glycines may serve as hinges enabling movement of the loops. In contrast, substitu tion of glycines at the N-terminal ends of beta-strands in the pla bar rel more often led to failure to detect L subunits or their assembly i nto L8S8 complex. Substitution of these and the other conserved glycin es by proline was more deleterious to carboxylase activity than by ala nine in enzymes that assembled.