Mutational analysis of residues in the regulatory CBS domains of Moorella thermoacetica pyrophosphatase corresponding to disease-related residues of human proteins.
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.
Department of Biochemistry and Food Chemistry, University of Turku, Finland.
Joonas
J
Jämsen
Heidi
H
Tuominen
Alexander A
AA
Baykov
Reijo
R
Lahti
D000249:Adenosine Monophosphate; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000235:genetics; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis; D004194:Disease / Q000235:genetics; D006801:Humans; D058845:Moorella / Q000201:enzymology* / Q000235:genetics; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000235:genetics; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D000249:Adenosine Monophosphate; D011755:Pyrophosphatases; D003541:Cystathionine beta-Synthase