title: Deuterium kinetic isotope effects in heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96: the anionic form of the substrate in the enzyme-substrate complex is a reactive species.
abstract: Heterotetrameric sarcosine oxidase is a flavoprotein that catalyses the oxidative demethylation of sarcosine. It is thought that the dehydrogenated substrate is the anionic form of sarcosine. To verify this assumption, the rate of flavin-adenine dinucleotide (FAD) reduction (k(red)) was analysed using protiated and deuterated sarcosine (N-methyl-d(3)-Gly) at various pH values using stopped-flow method. By increasing the pH from 6.2 to 9.8, k(red) increased for both substrates and reached a plateau, but the pK(a) value (reflecting the ionization of the enzyme-substrate complex) was 6.8 and 7.1 for protiated and deuterated sarcosine, respectively, and the kinetic isotope effect of k(red) decreased from approximately 19 to 8, indicating deprotonation of the bound sarcosine. The k(red)/K(d) (K(d), sarcosine dissociation constant) increased with increasing pH and reached a plateau. The pK (reflecting the ionization of free enzyme or free sarcosine) was 7.0 for both substrates, suggesting deprotonation of the βLys358 residue, which has a pK(a) of 6.7, as the pK(a) of the free sarcosine amine proton was determined to be approximately 10.1. These results indicate that the amine proton of sarcosine is transferred to the unprotonated Lys residue in the enzyme-substrate complex.
authors.affiliation: Division of Bioscience, Graduate School of Basic Life Science, Kitasato University, Kitasato, Sagamihara-shi, Japan.
authors.forename: Mutsumi
authors.initials: M
authors.lastname: Saito
authors.forename: Ai
authors.initials: A
authors.lastname: Itoh
authors.forename: Haruo
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D000838:Anions / Q000737:chemistry / Q000378:metabolism; D003352:Corynebacterium / Q000201:enzymology*; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050879:Sarcosine Oxidase / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000838:Anions; D003903:Deuterium; D050879:Sarcosine Oxidase