TY - JOUR
T1 - Nonessentiality of histidine 291 of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase as determined by site-directed mutagenesis
AU - Niyogi, S. K.
AU - Foote, R. S.
AU - Mural, R. J.
AU - Larimer, F. W.
AU - Mitra, S.
AU - Soper, T. S.
AU - Machanoff, R.
AU - Hartman, F. C.
PY - 1986
Y1 - 1986
N2 - Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B.A., and El-Gul, T., (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pK(a) of His-298 was observed to be ~6.8, and it was suggested that this histidine might be the essential base that inititates catalysis (Paech, C. (1985) Biochemistry 24, 3194-3199). To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the coresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is ~40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a k(cat) of 1.5 s-1 and a k(cat)/K(m) of 1.7·104 M-1 s-1 in contrast to values of 3.6 s-1 and 6·105 M-1 s-1 for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.
AB - Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B.A., and El-Gul, T., (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pK(a) of His-298 was observed to be ~6.8, and it was suggested that this histidine might be the essential base that inititates catalysis (Paech, C. (1985) Biochemistry 24, 3194-3199). To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the coresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is ~40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a k(cat) of 1.5 s-1 and a k(cat)/K(m) of 1.7·104 M-1 s-1 in contrast to values of 3.6 s-1 and 6·105 M-1 s-1 for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.
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M3 - Article
C2 - 3090029
AN - SCOPUS:0022854421
SN - 0021-9258
VL - 261
SP - 10087
EP - 10092
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 22
ER -