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Mutational analysis of residues in the regulatory CBS domains of Moorella thermoacetica pyrophosphatase corresponding to disease-related residues of human proteins
- Source :
- Biochemical Journal. 433:497-504
- Publication Year :
- 2011
- Publisher :
- Portland Press Ltd., 2011.
-
Abstract
- mtCBS-PPase [CBS (cystathionine β-synthase) domain-containing pyrophosphatase from Moorella thermoacetica] contains a pair of CBS domains that strongly bind adenine nucleotides, thereby regulating enzyme activity. Eight residues associated with the CBS domains of mtCBS-PPase were screened to explore possible associations with regulation of enzyme activity. The majority of the substitutions (V99A, R168A, Y169A, Y169F, Y188A and H189A) enhanced the catalytic activity of mtCBS-PPase, two substitutions (R170A and R187G) decreased activity, and one substitution (K100G) had no effect. AMP-binding affinity was markedly decreased in the V99A, R168A and Y169A mutant proteins, and elevated in the R187G and H189A mutant proteins. Remarkably, the R168A and Y169A substitutions changed the effect of AMP from inhibition to activation. The stoichiometry of AMP binding increased from one to two AMP molecules per CBS domain pair in the Y169F, R170A, R187G and Y188A variants. The ADP-binding affinity decreased in three and increased in four mutant proteins. These findings identify residues determining the strength and selectivity of nucleotide binding, as well as the direction (inhibition or activation) of the subsequent effect. The data suggest that mutations in human CBS domain-containing proteins can be translated into a bacterial context. Furthermore, our data support the hypothesis that the CBS domains act as an ‘internal inhibitor’ of mtCBS-PPase.
- Subjects :
- Amino Acid Motifs
DNA Mutational Analysis
Mutation, Missense
Cystathionine beta-Synthase
CBS domain
AMP binding
Biochemistry
03 medical and health sciences
chemistry.chemical_compound
Protein structure
Bacterial Proteins
Moorella thermoacetica
Adenine nucleotide
Moorella
Humans
Disease
Nucleotide
Pyrophosphatases
Molecular Biology
030304 developmental biology
chemistry.chemical_classification
0303 health sciences
Pyrophosphatase
biology
030302 biochemistry & molecular biology
Cell Biology
biology.organism_classification
Cystathionine beta synthase
Adenosine Monophosphate
Protein Structure, Tertiary
chemistry
biology.protein
Subjects
Details
- ISSN :
- 14708728 and 02646021
- Volume :
- 433
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal
- Accession number :
- edsair.doi.dedup.....b94885e589e91343e470b550c3040327