title: alpha-lactalbumin mutant acting as lysozyme.
abstract: A mutant of alpha-lactalbumin was expressed and purified, in which His32, Thr33, Glu49, Ile59, Val99, and Tyr103 were substituted by Leu32, Glu33, Asp49, Trp59, Asn99, and Ala103, respectively, to create a catalytic site of lysozyme in alpha-lactalbumin. The mutant catalyzed hydrolysis of the synthetic substrate, pNP-(NAcGlc)(3), with a K(M) and k(cat) of 0.160 +/- 0.00986 mmol/L and 3.39 +/- 0. 0456 x10(-5) min(-1), respectively, which was comparable with those of chicken lysozyme of 0.137 +/- 0.0153 mmol/L and 5.25 +/- 0.115 x10(-4) min(-1). By using the Isothermal Titration Calorimetre (ITC), the average binding enthalpy of the mutant or chicken lysozyme with the substrate (chitopentaose) was measured, which was 49.22 KJ/mol for the mutant and 105.47 KJ/mol for chicken lysozyme. In conclusion, the six point mutations occurring in alpha-lactalbumin could be converted into an enzyme that was 17.5-fold less efficient than chicken lysozyme but nevertheless capable of hydrolyzing the glycosidic bond.
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing, People's Republic of China.
authors.forename: Y
authors.initials: Y
authors.lastname: Xue
authors.forename: J N
authors.initials: JN
authors.lastname: Liu
authors.forename: Z
authors.initials: Z
authors.lastname: Sun
authors.forename: Z
authors.initials: Z
authors.lastname: Ma
authors.forename: C
authors.initials: C
authors.lastname: Wu
authors.forename: D
authors.initials: D
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002152:Calorimetry, Differential Scanning; D020134:Catalytic Domain; D002417:Cattle; D002645:Chickens; D019143:Evolution, Molecular; D006868:Hydrolysis; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016415:Sequence Alignment; D013379:Substrate Specificity; D013816:Thermodynamics; D014312:Trisaccharides / Q000378:metabolism
chemical_list: D014312:Trisaccharides; C041161:chitotriose; D007768:Lactalbumin; D009113:Muramidase