Kinetic studies of the mechanism of carbon-hydrogen bond breakage by the heterotetrameric sarcosine oxidase of Arthrobacter sp 1-IN

The reaction of heterotetrameric sarcosine oxidase (TSOX) of Arthrobactor s p. 1-IN has been studied by stopped-flow spectroscopy, with particular emph asis on the reduction of the enzyme by sarcosine. Expression of the cloned gene encoding TSOX in Escherichia coli enables the production of TSOX on a scale suitable for stopped-flow studies. Treatment of the enzyme with sulfi te provides the means for selective formation of a flavin-sulfite adduct wi th the covalent 8 alpha-(N-3-histidyl)-FMN. Formation of the sulfite-flavin adduct suppresses internal electron transfer between the noncovalent FAD ( site of sarcosine oxidation) and the covalent FMN (site of enzyme oxidation ) and thus enables detailed characterization of the kinetics of FAD reducti on by sarcosine using stopped-flow methods. The rate of FAD reduction displ ays a simple hyperbolic dependence on sarcosine concentration. Studies in t he pH range 6.5-10 indicate there are no kinetically influential ionization s in the enzyme-substrate complex. A plot of the limiting rate of flavin re duction/the enzyme-substrate dissociation constant (k(lim)/K-d) versus pH i s bell-shaped and characterized by two macroscopic pK(a) values of 7.4 +/- 0.1 and 10.4 +/- 0.2. potential candidates for the two ionizable groups are discussed with reference to the structure of monomeric sarcosine oxidase ( MSOX). The kinetic data are discussed with reference to potential mechanism s for the oxidation of amine molecules by flavoenzymes. Additionally, kinet ic isotope effect studies of the rate of C-H bond breakage suggest that a g round-state quantum tunneling mechanism for H-transfer, facilitated by the low-frequency thermal motions of the protein molecule, accounts for C-H bon d cleavage by TSOX. TSOX thus provides another example of C-H bond breakage by ground-state quantum tunneling, driven by protein dynamics [vibrational ly enhanced ground-state quantum tunneling (VEGST)], for the oxidation of a mines by enzymes.