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H B Smith

Publications and source records attributed to H B Smith.

11 recordsLinked to original sources
PubMed1991

Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue.

Ribulosebisphosphate carboxylase/oxygenase is reversibly activated by the reaction of CO2 with a specific lysyl residue (Lys191 of the Rhodospirillum rubrum enzyme) to form a carbamate that coordinates an essential Mg2+ cation. Surprisingly, the Lys191----Cys mutant protein, in the presence of CO2 and Mg2+, exhibits tight binding of the reaction intermediate analogue 2-carboxyarabinitol bisphosphate [Smith, H. B., Larimer, F. W., & Hartman, F. C. (1988) Biochem. Biophys. Res. Commun. 152, 579-584], a property normally equated with effective coordination of the Mg2+ by the carbamate. Catalytic ineptness of the Cys191 mutant protein, despite its ability to coordinate Mg2+ properly, might be due to the absence of the carbamate nitrogen. To investigate this possibility, we have evaluated the ability of exogenous amines to restore catalytic activity to the mutant protein. Significantly, the Cys191 protein manifests ribulose bisphosphate dependent fixation of 14CO2 when incubated with aminomethanesulfonate but not ethanesulfonate. This novel activity reflects a Km value for ribulose bisphosphate which is not markedly perturbed relative to wild-type enzyme, a Km for Mg2+ which is in fact decreased 10-fold, and rate saturation with respect to aminomethanesulfonate (Kd = 8 mM). Chromatographic and spectrophotometric analyses reveal the product of CO2 fixation to be D-3-phosphoglycerate, while turnover of [1-3H]ribulose bisphosphate into [3H]phosphoglycolate confirms oxygenase activity. We conclude that aminomethanesulfonate restored ribulosebisphosphate carboxylase/oxygenase activities to the Cys191 mutant protein by providing a nitrogenous function which satisfies a catalytic demand normally met by the carbamate nitrogen of Lys191.

Binding Sites↗
PubMed1990

An engineered change in substrate specificity of ribulosebisphosphate carboxylase/oxygenase.

The potential for altering the specificity of ribulosebisphosphate carboxylase/oxygenase toward gaseous substrates is explored through a modest perturbation of the active site microenvironment. Specifically, replacement of active site Glu-48 with carboxy-methylcysteine is achieved in a two-step process in which the catalytically incompetent Cys-48 mutant protein is first generated and then treated with iodoacetic acid. This regimen of concerted site-directed mutagenesis and chemical modification, effectively lengthening the glutamyl side chain by insertion of a sulfur atom between the beta- and gamma-methylene groups, results in a protein possessing 4-6% of wild-type carboxylase activity. Concomitantly, the engineered enzyme exhibits a specificity factor 5-fold lower than that of wild-type enzyme. This represents the first example of a major change in substrate specificity, albeit in favor of oxygenation, effected by structural alteration of an active site side chain.

Binding Sites↗
PubMed1988

Subtle alteration of the active site of ribulose bisphosphate carboxylase/oxygenase by concerted site-directed mutagenesis and chemical modification.

Both activities of ribulose bisphosphate carboxylase/oxygenase are dependent on carbamylation by CO2 of a specific lysyl epsilon-amino group (Lys-191 of the enzyme from Rhodospirillum rubrum). To examine the stringency of the requirement for this lysyl side chain, Lys-191 was converted to an aminoethylcysteinyl residue (net replacement of a gamma-methylene group by a sulfur atom) by a combination of site-directed mutagenesis and subsequent chemical modification. The purified Cys-191 mutant was totally devoid of both carboxylase and oxygenase activities. However, this mutant protein exhibited tight-binding of the transition-state analogue, 2-carboxyarabinitol bisphosphate, a property heretofore ascribed solely to the carbamylated form of the carboxylase. Treatment of the mutant protein with ethylene imine restored catalytic activity to 4-7% of the wild-type level. The carboxylase:oxygenase activity ratio of the aminoethylated protein was unperturbed relative to that of wild-type enzyme.

Amino Acid Sequence↗
PubMed1988

Restoration of activity to catalytically deficient mutants of ribulosebisphosphate carboxylase/oxygenase by aminoethylation.

Substitutions for active-site lysyl residues at positions 166 and 329 in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been shown to abolish catalytic activity. Treatment of the Cys-166 and Cys-329 mutant proteins with 2-bromoethylamine partially restores enzyme activity, presumably as a consequence of selective aminoethylation of the thiol group unique to each protein. Amino acid analyses, slow inactivation of the wild-type carboxylase by bromoethylamine, and the failure of bromoethylamine to restore activity to the corresponding glycyl mutant proteins support this interpretation. The observed facile, selective aminoethylations may reflect an active site microenvironment not dissimilar to that of the native enzyme. Catalytic constants of these novel carboxylases, which contain a sulfur atom in place of a specific lysyl gamma-methylene group, are significantly lower than that of the wild-type enzyme. Furthermore, the aminoethylated mutant proteins form isolable complexes with a transition state analogue, but with compromised stabilities. These detrimental effects by such a modest structural change underscore the stringent requirement for lysyl side chains at positions 166 and 329. In contrast, the aminoethylated mutant proteins exhibit carboxylase/oxygenase activity ratios and Km values that are unperturbed relative to those for the native enzyme.

Binding Sites↗
PubMed1987

Dental management of patients with sickle cell disorders.

The pathogenesis, clinical characteristics, screening protocol, and dental management of the patient with sickle cell anemia or sickle cell trait are discussed. Treatment modalities are presented to aid the practitioner in caring for the patient with a sickle cell disorder.

Anemia, Sickle Cell↗