Monomeric Sarcosine Oxidase:  2. Kinetic Studies with Sarcosine, Alternate Substrates, and a Substrate Analogue

Abstract

Monomeric sarcosine oxidase (MSOX) is a flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) to yield glycine, formaldehyde, and hydrogen peroxide. MSOX can oxidize other secondary amino acids (N-methyl-L-alanine, N-ethylglycine, and L-proline), but N,N-dimethylglycine, a tertiary amine, is not a substrate. N-Methyl-L-alanine is a good alternate substrate, exhibiting a k(cat) value (8700 min(-)(1)) similar to sarcosine (7030 min(-)(1)). Turnover with L-proline (k(cat) = 25 min(-)(1)) at 25 degrees C occurs at less than 1% of the rate observed with sarcosine. MSOX is converted to a two-electron reduced form upon anaerobic reduction with sarcosine or L-proline. No evidence for a spectrally detectable intermediate was obtained in reductive half-reaction studies with L-proline. The reductive half-reaction with L-proline at 4 degrees C exhibited saturation kinetics (k(lim) = 6.0 min(-)(1), K(d) = 260 mM) and other features consistent with a mechanism in which a practically irreversible reduction step (E(ox). S --> E(red).P) with a rate constant, k(lim), is preceded by a rapidly attained equilibrium (K(d)) between free E and the E.S complex. Steady-state kinetic studies with sarcosine and N-methyl-L-alanine in the absence or presence of a dead-end inhibitor (pyrrole-2-carboxylate) indicate that catalysis proceeds via a "modified" ping pong mechanism in which oxygen reacts with E(red).P prior to the dissociation of the imino acid product. In this mechanism, double reciprocal plots will appear nearly parallel (as observed) if the reduction step is nearly irreversible. A polar mechanism, involving formation of a covalent 4a-flavin-substrate adduct is one of several plausible mechanisms for sarcosine oxidation. Thiols are known to form similar 4a-flavin adducts. MSOX does not form a 4a-adduct with thioglycolate but does form a charge-transfer complex that undergoes an unanticipated one-electron-transfer reaction to yield the anionic flavin radical.

Involvement of Ionizable Groups in Catalysis of Human Liver Glycolate Oxidase

“…This conclusion is supported by the pH profile of the second-order rate constant k cat /K oxygen determined under steady-state kinetics conditions, which increased from a limiting value of ϳ7,000 M Ϫ1 s Ϫ1 at low pH to a limiting value of ϳ60,000 M Ϫ1 s Ϫ1 at high pH values. The limiting value in the upper 10 4 M Ϫ1 s Ϫ1 range seen at high pH is in keeping with values previously reported for other flavoprotein oxidases, such as, for example, spinach GOX (17), choline oxidase (43,44), monoamine oxidase (45,46), nitroalkane oxidase (47), lactate oxidase (48), sarcosine oxidase (49), and glucose oxidase (50), and it is ϳ2.5 orders of magnitude larger than the value of 2.5 ϫ 10 2 M Ϫ1 s Ϫ1 that was reported for the non-enzymatic oxidation of flavins in solutions (48,51). However, the pH profile for the k cat /K oxygen value for GOX is significantly different from the pH profiles of the k cat /K oxygen values that were previously reported for glucose oxidase and choline oxidase.…”

Structure-Function Correlation in Glycine Oxidase from Bacillus subtilis

“…GO follows a ternary complex sequential mechanism with glycine, sarcosine, and D-proline as substrates in which the rate of product dissociation from the re-oxidized enzyme form represents the rate-limiting step (5). Such a kinetic mechanism is similar to that determined for mammalian DAAO on neutral D-amino acids and for the MSOX on L-proline (6,7); the main difference is represented by the observed reversibility of the GO reductive half-reaction.…”

Purification and Properties of Glycine Oxidase from <i>Pseudomonas putida</i> KT2440

“…As shown in Fig. 3, GO homologs appeared to constitute a diverse group of flavoenzymes, which are distantly related to monomeric sarcosine oxidase (14) and dye-dependent d-amino acid dehydrogenases, such as d-alanine dehydrogenase (15), d-arginine dehydrogenase (16), and d-proline dehydrogenase (17). The analysis resolved the GO homologs into three distinct clades (the betaproteobacteria/gammaproteobacteria clade, the bacilli/alphaproteobacteria/deltaproteobacteria clade, and the planctomycetia clade).…”