Effect of Cysteine Residue Substitution in the GCSAG Motif of the PMGL2 Esterase Active Site on the Enzyme Properties

Autor:	D. A. Dolgikh, Alena Yu. Nikolaeva, Konstantin M. Boyko, E. A. Kryukova, S. A. Yakimov, K. A. Novototskaya-Vlasova, Mikhail P. Kirpichnikov, Lada E. Petrovskaya, M.V. Kryukova
Rok vydání:	2020
Předmět:	Models Molecular Mutant Biophysics Permafrost Biochemistry Biochemistry Genetics and Molecular Biology (miscellaneous) Esterase Substrate Specificity Serine 03 medical and health sciences Catalytic Domain Cysteine Enzyme kinetics Enzyme Assays chemistry.chemical_classification 0303 health sciences biology Chemistry 030302 biochemistry & molecular biology Esterases Active site General Medicine Sterol Esterase Kinetics Enzyme Mutation Mutagenesis Site-Directed biology.protein Specific activity Geriatrics and Gerontology
Zdroj:	Biochemistry (Moscow). 85:709-716
ISSN:	1608-3040 0006-2979
Popis:	The gene coding for PMGL2 esterase, which belongs to the family of mammalian hormone-sensitive lipases (HSLs), was discovered by screening a metagenomic DNA library from a permafrost soil. The active site of PMGL2 contains conserved GXSXG motif which includes Cys173 residue next to the catalytic Ser174. In order to clarify the functional role of the cysteine residue in the GCSAG motif, we constructed a number of PMGL2 mutants with Cys173 substitutions and studied their properties. The specific activity of the C173D mutant exceeded the specific activity of the wild-type enzyme (wtPMGL2) by 60%, while the C173T/C202S mutant displayed reduced catalytic activity. The activity of the C173D mutant with p-nitrophenyl octanoate was 15% higher, while the activity of the C173T/C202S mutant was 17% lower compared to wtPMGL2. The C173D mutant was also characterized by a high activity at low temperatures (20-35°C) and significant loss of thermal stability. The kcat value for this protein was 56% higher than for the wild-type enzyme. The catalytic constants of the C173S mutant were close to those of wtPMGL2; this enzyme also demonstrated the highest thermal stability among the studied mutants. The obtained results demonstrate that substitutions of amino acid residues adjacent to the catalytic serine residue in the GXSXG motif can have a significant effect on the properties of PMGL2 esterase.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e722b197ba65b79eb1131791231db7fd https://doi.org/10.1134/s0006297920060085 Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
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