Role of phenylalanine-327 in the closure of loop 6 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

Phenylalanine-327 of ribulosebisphosphate carboxylase/oxygenase (rubisco) from Rhodospirillum rubrum was mutated to tryptophan, leucine, valine, alanine, and glycine, and was also deleted. The least active mutant, the deletion mutant, exhibits less than 0.5% of the carboxylase activity of the wild-type enzyme. Steady-state kinetic analysis of F327-->Leu, Val, Ala, Gly mutant enzymes reveals that kcat and the CO2/O2 specificity are unchanged while Km(RuBP) (RuBP = ribulose 1,5-bisphosphate) is drastically increased. The mutant enzyme with the highest value for Km(RuBP),Phe327-->Gly, shows a 165-fold increase (1160 microM compared to 7 microM for the wild-type). The increase in Km(RuBP) suggests an alteration of the ratio kon/koff for RuBP. A longer hydrophobic lateral chain and/or the presence of an aromatic ring in the wild-type enzyme and the Phe327-->Trp mutant enzyme could explain a better packing of loop 6 in the closed conformation and thus a tighter binding of RuBP at the active site.

[1]  A. Fersht,et al.  Mutation of asparagine 111 of rubisco from Rhodospirillum rubrum alters the carboxylase/oxygenase specificity. , 1992, Journal of molecular biology.

[2]  Xiang-Jiao Yang,et al.  Threonine 183 and adjacent flexible loop residues in the tryptophan synthase alpha subunit have critical roles in modulating the enzymatic activities of the beta subunit in the alpha 2 beta 2 complex. , 1992, The Journal of biological chemistry.

[3]  H. Kato,et al.  Mutational and proteolytic studies on a flexible loop in glutathione synthetase from Escherichia coli B: the loop and arginine 233 are critical for the catalytic reaction. , 1992, Biochemistry.

[4]  R. J. Spreitzer,et al.  Complementing amino acid substitutions within loop 6 of the alpha/beta-barrel active site influence the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. , 1991, Biochemistry.

[5]  S. Gutteridge.  The relative catalytic specificities of the large subunit core of Synechococcus ribulose bisphosphate carboxylase/oxygenase. , 1991, The Journal of biological chemistry.

[6]  J. Knowles,et al.  Enzyme catalysis: not different, just better , 1991, Nature.

[7]  G. Schneider,et al.  Crystal structure of the ternary complex of ribulose-1,5-bisphosphate carboxylase, Mg(II), and activator CO2 at 2.3-A resolution. , 1991, Biochemistry.

[8]  M Karplus,et al.  Anatomy of a conformational change: hinged "lid" motion of the triosephosphate isomerase loop. , 1990, Science.

[9]  I. Andersson,et al.  Crystallographic analysis of ribulose 1,5-bisphosphate carboxylase from spinach at 2.4 A resolution. Subunit interactions and active site. , 1990, Journal of molecular biology.

[10]  Gregory A. Petsko,et al.  The evolution of a/ barrel enzymes , 1990 .

[11]  J. Knowles,et al.  Stabilization of a reaction intermediate as a catalytic device: definition of the functional role of the flexible loop in triosephosphate isomerase. , 1990, Biochemistry.

[12]  G. Schneider,et al.  Crystallographic refinement and structure of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum at 1.7 A resolution. , 1990, Journal of molecular biology.

[13]  J. Berry,et al.  Radiochemical Assay of Ribulose Bisphosphate Carboxylase , 1989 .

[14]  M. Parry,et al.  Variation in the Specificity Factor of C3 Higher Plant Rubiscos Determined by the Total Consumption of Ribulose-P2 , 1989 .

[15]  R. J. Spreitzer,et al.  Chloroplast intragenic suppression enhances the low CO2/O2 specificity of mutant ribulose-bisphosphate carboxylase/oxygenase. , 1989, The Journal of biological chemistry.

[16]  F. C. Hartman,et al.  Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis. , 1988, Protein engineering.

[17]  A. Fersht,et al.  Reconstruction by site-directed mutagenesis of the transition state for the activation of tyrosine by the tyrosyl-tRNA synthetase: a mobile loop envelopes the transition state in an induced-fit mechanism. , 1988, Biochemistry.

[18]  D. Hill,et al.  Ribulose 1,5-bisphosphate carboxylase. Effect on the catalytic properties of changing methionine-330 to leucine in the Rhodospirillum rubrum enzyme. , 1986, The Biochemical journal.

[19]  A. Mildvan,et al.  ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Alan R. Fersht,et al.  The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus) , 1984, Cell.

[21]  J. Pierce,et al.  Interaction of ribulosebisphosphate carboxylase/oxygenase with transition-state analogues. , 1980, Biochemistry.

[22]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[23]  G. Schneider,et al.  Crystal structure of the ternary complex of ribulose-1,5-bisphosphate carboxylase, magnesium(II) and activator carbon dioxide at 2.3-.ANG. resolution , 1991 .

[24]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.