Spectral and Kinetic Characterization of the Michaelis Charge Transfer Complex in Monomeric Sarcosine Oxidase

Abstract

Monomeric sarcosine oxidase is a flavoenzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine). Rapid reaction kinetic studies under anaerobic conditions at pH 8.0 show that the enzyme forms a charge transfer Michaelis complex with sarcosine (E-FAD(ox).sarcosine) that exhibits an intense long-wavelength absorption band (lambda(max) = 516 nm, epsilon(516) = 4800 M(-)(1) cm(-)(1)). Since charge transfer interaction with sarcosine as donor is possible only with the anionic form of the amino acid, the results indicate that the pK(a) of enzyme-bound sarcosine must be considerably lower than the free amino acid (pK(a) = 10.0). No redox intermediate is detectable during sarcosine oxidation, as judged by the isosbestic spectral course observed for conversion of E-FAD(ox).sarcosine to reduced enzyme at 25 or 5 degrees C. The limiting rate of the reductive half-reaction at 25 degrees C (140 +/- 3 s(-)(1)) is slightly faster than turnover (117 +/- 3 s(-)(1)). The kinetics of formation of the Michaelis charge transfer complex can be directly monitored at 5 degrees C where the reduction rate is 4.5-fold slower and complex stability is increased 2-fold. The observed rate of complex formation exhibits a hyperbolic dependence on sarcosine concentration with a finite Y-intercept, consistent with a mechanism involving formation of an initial complex followed by isomerization to yield a more stable complex. Similar results are obtained for charge transfer complex formation with methylthioacetate. The observed kinetics are consistent with structural studies which show that a conformational change occurs upon binding of methylthioacetate and other competitive inhibitors.

Identification of the Oxygen Activation Site in Monomeric Sarcosine Oxidase: Role of Lys265 in Catalysis

“…This feature suggested that the reductive half-reaction with Lys265Met is initiated by the formation of an enzyme•substrate charge transfer complex, similar to that observed with wild-type MSOX (12). Consistent with this hypothesis, initial spectra observed after mixing Lys265Met with a wide range of sarcosine concentrations (1 to 140 mM) are isosbestic (Figure 5A) and show the progressive formation of a charge transfer band that exhibits a maximum at 495 nm, as estimated by the position of the peak in the corresponding difference spectrum (Figure 6A).…”

“…An isosbestic spectral course is observed for the anaerobic reduction of wild-type MSOX over a wide range of sarcosine concentrations (1 to 80 mM) in reactions that exhibit a monophasic exponential loss of oxidized enzyme absorbance (12). Apparently similar features are observed for the reductive half-reactions with Lys265Met or Lys265Arg but only at sarcosine concentrations below 25 or 10 mM, respectively (data not shown, see Supporting Information Figure S1).…”

“…Dissociation constants for the complex formed with Lys265Met or Lys265Arg and methylthioacetate were determined by fitting a theoretical binding curve to absorbance changes observed at 515 or 518 nm, respectively. Spectra corresponding to 100% complex formation were calculated as previously described (12). Special cuvettes with two side arms were used for anaerobic experiments.…”

“…Dissociation constants for the complexes were determined by fitting a theoretical binding curve to initial absorbance changes observed at 495 or 497 nm, respectively. Spectra corresponding to 100% complex formation were calculated as previously described (12). Fitting of single-wavelength kinetics traces were conducted using Sigma Plot 10 (Systat Software).…”

“…Access to the active site is controlled by a loop (Gly56 to Glu60) that forms part of a cleft leading from the surface. The active site loop is mobile in the free enzyme but assumes a closed conformation in enzyme complexes with competitive inhibitors (6, 11, 12). …”