scholarly journals Study of the Interaction of Sorption and Catalytic Centers in Carboxypeptidase T by X-ray Analysis

Crystals ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1088
Author(s):  
Valerij Akparov ◽  
Vladimir Timofeev ◽  
Inna Kuranova ◽  
Ilias Khaliullin
Keyword(s):  
Substrate Specificity ◽  
Transition State ◽  
Kinetic Data ◽  
Carboxypeptidase B ◽  
Catalytic Center ◽  
X Ray ◽  
Thermoactinomyces Vulgaris ◽  
Distant Homolog ◽  
Carboxypeptidase T ◽  
Primary Specificity

Carboxypeptidase T (CPT; EC 3.4.17.18) from Thermoactinomyces vulgaris is a distant homolog of the highly specific pancreatic carboxypeptidase B; but has a broad substrate specificity; the source of which remains unclear. A previous study of the structural bases of the substrate specificity of CPT using stable sulfamoyl analogs of the transition state of the elimination of leucine; phenylalanine; arginine; and glutamic acid; showed that the binding of the C-terminal residue of the substrate to the primary selectivity pocket of CPT leads to a change in the distance between Zn2+ and the sulfur atom. This value is related to the efficiency of catalysis of the corresponding substrate or the inhibition constant of the corresponding stable analog of the transition state. In this work; we obtained crystallographic and kinetic data of the complex of CPT with N-sulfamoyl-L-valine; confirming the effect of the binding of the ligand’s side group by the primary specificity pocket of CPT on the structure of the catalytic center; which can explain the unusual substrate specificity of CPT.

Molecular cloning and primary structure of Thermoactinomyces vulgaris carboxypeptidase T A metalloenzyme endowed with dual substrate specificity

FEBS Letters ◽  
1991 ◽  
Vol 291 (1) ◽  
pp. 75-78 ◽  
Author(s):  
Sergey V. Smulevitch ◽  
Andrey L. Osterman ◽  
Olga V. Galperina ◽  
Mikhail V. Matz ◽  
Olga P. Zagnitko ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Molecular Cloning ◽  
Primary Structure ◽  
Thermoactinomyces Vulgaris ◽  
Dual Substrate Specificity ◽  
Carboxypeptidase T ◽  
Dual Substrate

scholarly journals Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1′ subsite from pancreatic carboxypeptidase B

2018 ◽  
Vol 74 (10) ◽  
pp. 638-643 ◽  
Author(s):  
Valery Kh. Akparov ◽  
Vladimir I. Timofeev ◽  
Inna P. Kuranova ◽  
Tatiana V. Rakitina
Keyword(s):  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Carboxypeptidase B ◽  
X Ray Crystallography ◽  
Thermoactinomyces Vulgaris ◽  
The Difference ◽  
A Site ◽  
Carboxypeptidase T

A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1′ subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1′ subsite was crystallized and its three-dimensional structure was determined at 1.29 Å resolution by X-ray crystallography. A comparison of the three-dimensional structures of CPT, the G215S/A251G/T257A/D260G/T262D CPT mutant and CPB showed that the S1′ subsite of CPT has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1′ subsite. The CPB-like mutant differs from CPB in substrate selectivity owing to differences between the two enzymes outside the S1′ subsite. Moreover, the difference in substrate specificity between the enzymes was shown to be affected by residues other than those that directly contact the substrate.

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