Reaction of Formaldehyde and of Methanol with Xanthine Oxidase

Abstract

Earlier studies on inactivation of xanthine oxidase induced by methanol and accompanied by development of a specific electron paramagnetic resonance signal (the Inhibited signal) have been extended. The same reaction takes place when enzyme is treated with formaldehyde. With this, time-courses for signal development and disappearance of enzymic activity cannot be distinguished. Under a variety of conditions, disappearance of the signal accompanied by partial restoration of activity could be achieved. Side reactions also take place in the presence of formaldehyde. Electron paramagnetic resonance parameters of the Inhibited signal are reported. I n the signal-giving species a single non-exchangeable proton interacts with Mo(V) in the enzyme active site. Experiments with C2H30H and with 2HC2H0 show that this proton is derived from the inactivating agent. It is proposed that a formyl residue, -CHO, becomes attached to a group in the active centre and that interaction of molybdenum with this residue stabilizes the five-valent state of the metal, thus preventing enzyme turnover. Methanol is presumed to be oxidized to formaldehyde a t the active centre before taking part in the reaction.Studies on the interaction of enzymes with specific irreversible inhibitors may yield information important in understanding their catalytic mechanisms. Specific inhibition of xanthine oxidase by methanol seems first to have been noted by Polonovski [l]. More detailed studies were carried out by Handler and co-workers (e.g. [2,3]), who found that inhibition develops progressively and only while the enzyme is turning over in the presence of substrates plus methanol. They proposed [4] that this somewhat unusual inhibition involves a molecule of methanol substituting for one of water in the coordination sphere of molybdenum in the active site of the enzyme. They further reported [4] tion [6]) that the doublets were replaced by single lines, establishing the interacting proton t o be derived from the methyl group of methanol. We now report further studies carried out with the aim of understanding the reaction between milk xanthine oxidase and methanol.

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

“…25). Ethylene glycol is a slow substrate of xanthine oxidase (26) and EPR measurements of xanthine oxidase inhibited with methanol and formaldehyde yield identical "inhibited" signals (27) in accord with similar binding modes for alcohols and aldehydes. Specifically, substrate binding in the second coordination sphere of the molybdenum in the Michaelis complex is supported by the absence in xanthine oxidase of proton coupling in the "rapid type 1" EPR signal due to the C-8-bound proton of the slowly reacting substrate 2-hydroxy-6-methylpurine (28) (coupling occurs only after substrate oxidation and hydride transfer), by the interaction of 8-bromoxanthine (a strong inhibitor of xanthine oxidase) in a way that is typical of purine substrates (29) with a Mo-Br distance greater than 4 A (30), and by the binding of arsenite to the metal which forms a first coordination sphere complex without interfering with binding of substrates (31).…”

Spectroscopic and Electronic Structure Studies Probing Covalency Contributions to C–H Bond Activation and Transition-State Stabilization in Xanthine Oxidase

“…Formaldehyde Inhibited XO displays a strongly coupled proton 48,49 that is not evident in the 3-pyridinecarboxaldehyde spectra, and this indicates that the aldehydic R group is oriented in the “up” or M≡O direction (Figure 2). A previous EPR study of Inhibited utilized a 95,97 Mo isotope perturbation to obtain the hyperfine parameters $A_{\mathit{iso}}^{Mo}=42.72\times {10}^{-4}{\text{cm}}^{-1}$ and ${\mathbf{A}}_{\mathrm{boldS}}^{\mathbf{Mo}}=[+9.83,-19.67,+9.83]\times {10}^{-4}{\text{cm}}^{-1}$.…”

Structure and function of the xanthine-oxidase family of molybdenum enzymes

“…A number of aldehydes are found to inactivate bovine milk xanthine oxidase [92], while reactivation occurs immediately after removing excess aldehyde. EPR data from xanthine oxidase inhibited with methanol and formaldehyde produced identical "inhibited" signals [95], which suggests similar binding modes for alcohols and aldehydes. The binding of substrate molecules in the second coordination sphere of molybdenum in the Michaelis complex is also supported by spectroscopic data: In the reaction of xanthine oxidase with 2-hydroxy-6-methylpurine (a slowly reacting substrate), no proton coupling due to the C8-H proton is detected in the "rapid type 1" EPR signal [96], although coupling is detected after substrate oxidation and hydride transfer [50,96].…”