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 Anacystis nidulans by directed mutagenesis is described. In each beta/alpha barrel of the large subunit there are 12 completely conserved glycines in six of eight loops at the C-termini of eight beta-strands and four in loops at N-terminal ends of the beta-strands. Two completely conserved glycines are also in each beta/alpha barrel backbone and four more are in a large N-terminal portion preceding the barrel in a given L subunit. Substitution of glycines in loops that are C-terminal to beta-strands by proline was more deleterious to carboxylase activity than that by alanine supporting the postulates that these loops contribute to catalysis and substrate binding and that in some cases the glycines may serve as hinges enabling movement of the loops. In contrast, substitution of glycines at the N-terminal ends of beta-strands in the beta/alpha barrel more often led to failure to detect L subunits or their assembly into L8S8 complex. Substitution of these and the other conserved glycines by proline was more deleterious to carboxylase activity than by alanine in enzymes that assembled.
Cheng
Z Q
ZQ
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
McFadden
B A
BA
D000409:Alanine; D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D017124:Conserved Sequence; D000458:Cyanobacteria / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D011392:Proline; D012273:Ribulose-Bisphosphate Carboxylase; D000409:Alanine; D005998:Glycine