Leu254 residue and calcium ions as new structural determinants of carboxypeptidase T substrate specificity.
New determinants of Thermoactinomyces vulgaris carboxypeptidase T (CPT) substrate specificity--structural calcium ions and Leu254 residue--were found by means of steady-state kinetics and site-directed mutagenesis. The removal of calcium ions shifted the selectivity profile of hydrolysis of tripeptide substrates with C-terminal Leu, Glu, and Arg from 64/1.7/1 to 162/1.3/1. Substitution of the hydrophobic Leu254 in CPT for polar Asn did not change hydrolysis efficiency of substrates with C-terminal Leu and Arg, but resulted in more than 28-fold decrease in activity towards the substrate with C-terminal Glu. It is shown that the His68 residue is not a structural determinant of CPT specificity.
Grishin
A M
AM
Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 117545, Russia.
Akparov
V Kh
VKh
Chestukhina
G G
GG
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002268:Carboxypeptidases; C076614:CPT protein, Thermoactinomyces vulgaris; D007930:Leucine; D002118:Calcium