Kinetic Studies of the Mechanism of Carbon−Hydrogen Bond Breakage by the Heterotetrameric Sarcosine Oxidase of Arthrobacter sp. 1-IN

Abstract

The reaction of heterotetrameric sarcosine oxidase (TSOX) of Arthrobactor sp. 1-IN has been studied by stopped-flow spectroscopy, with particular emphasis 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 sulfite provides the means for selective formation of a flavin-sulfite adduct with the covalent 8alpha-(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 reduction by sarcosine using stopped-flow methods. The rate of FAD reduction displays a simple hyperbolic dependence on sarcosine concentration. Studies in the pH range 6.5-10 indicate there are no kinetically influential ionizations in the enzyme-substrate complex. A plot of the limiting rate of flavin reduction/the enzyme-substrate dissociation constant (k(lim)/K(d)) versus pH is 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 mechanisms for the oxidation of amine molecules by flavoenzymes. Additionally, kinetic isotope effect studies of the rate of C-H bond breakage suggest that a ground-state quantum tunneling mechanism for H-transfer, facilitated by the low-frequency thermal motions of the protein molecule, accounts for C-H bond cleavage by TSOX. TSOX thus provides another example of C-H bond breakage by ground-state quantum tunneling, driven by protein dynamics [vibrationally enhanced ground-state quantum tunneling (VEGST)], for the oxidation of amines by enzymes.

Linking protein structure and dynamics to catalysis: the role of hydrogen tunnelling

“…As observed initially in the reaction catalysed by soybean lipoxygenase (SLO; Glickman et al 1994;Hwang & Grissom 1994) and, subsequently, in a wide range of other enzyme-catalysed C-H abstraction reactions (e.g. Basran et al 1999Basran et al , 2001Kohen et al 1999;Abad et al 2000;Harris et al 2000;Francisco et al 2002;Sikorski et al 2004), the value of A 1 /A 2 often lies considerably above unity. In one case (dihydrofolate reductase, (Sikorski et al 2004)), a complex set of conditions have been postulated to explain A 1 /A 2 [1 within the tenets of variational transition state theory (VTST; Pu et al 2005).…”

Dynamic barriers and tunneling. New views of hydrogen transfer in enzyme reactions

“…In the last four years, a growing number of enzyme systems have been demonstrated to have properties that simply cannot be fit by the Bell correction [11,[17][18][19][20][21][22]. These are summarized in Table 1 where in most systems, significant effort has been expended to demonstrate that H-transfer is rate-limiting in the experimental temperature range.…”

Convex Arrhenius plots and their interpretation

“…1). Some of the methodologies for measuring the temperature dependences of reaction rates have been applied to measure the temperature dependences of the kinetic isotope effects (KIEs) of the catalyzed reactions as well, and such experiments provide experimental data very relevant to characterizing the dynamical bottlenecks of the reactions (1)(2)(3)(4)(5)(6). A large number of theoretical models have been advanced for the interpretation of this kind of data (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).…”