Primary structure of carboxypeptidase T: delineation of functionally relevant features in Zn-carboxypeptidase family.
The primary structure of carboxypeptidase T--a Zn-dependent extracellular enzyme of Thermoactinomyces vulgaris--was determined from the cloned cpT gene nucleotide sequence and compared to Zn-carboxypeptidases from various organisms. The compilation and analysis of multiple alignment accompanied by consideration of available tertiary structure data have shown that in the overall spatial structure and active site arrangement CpT is similar to other enzymes constituting the Zn-carboxypeptidase family. Nine of 16 amino acid residues found to be strictly invariant are presumably located close to the active site. The preservation of His69, Glu72, Asn144, Arg145, His196, Tyr248, and Glu270 identified previously as essential catalytic site participants implicates basically the same catalytic mechanism in the Zn-carboxypeptidase family. It is proposed that Pro205 and Asp256 should play an important role in proper S1'-pocket spatial arrangement. The comparative analysis of amino acid variations in S1'-pocket enabled us to reveal structural determinants of the Zn-carboxypeptidase primary specificity. The relatively reduced size of the pocket and negative charge of Asp253 are supposed to contribute correspondingly to A- and B-type substrate preferences of carboxypeptidase T endowed with dual primary specificity.
Osterman
A L
AL
Laboratory of Protein Chemistry, Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
Grishin
N V
NV
Smulevitch
S V
SV
Matz
M V
MV
Zagnitko
O P
OP
Revina
L P
LP
Stepanov
V M
VM
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology*
D001426:Bacterial Proteins; D002268:Carboxypeptidases; C076614:CPT protein, Thermoactinomyces vulgaris; D015032:Zinc