Flavoprotein oxidase kinetics

R. Hille and R.F. Anderson, Coupled electron/proton transfer in complex flavoproteins — solvent kinetic isotope effect studies of electron transfer in xanthine oxidase and trimethylamine dehydrogenase. J. Biol. Chem. 276, 31193-31201 (2001). 

This chapter is not comprehensive since it reflects the interest of the author in the kinetic and chemical mechanism of flavoprotein oxidase reactions. It is meant to complement the special interests of others who have written excellent reviews from their respective view points. The... 

The flavin coenzyme occurs in each of the oxidases discussed here as flavin adenine dinucleotide (FAD). The R group attached to is adenosyldiphosphoribityl. The flavin nucleus can exist in three redox states, each of which can adopt three ionization states (8, 9). Only two redox states—fully oxidized and fully reduced see Equation 1)—are kinetically important in the simple flavoprotein oxidases under discussion. 

Fortunately, the characteristic absorbance of certain stable and transient enzyme species and, in some instances, of products, together with the fact that the two half-reactions can be studied separately, permits informative rapid kinetic measurements of the overall and partial reactions of flavoprotein oxidases. Stopped-flow spectrophotometric methods (26) have been particularly useful (the irreversibility of the partial and overall reactions rules out relaxation methods) because the measured rate constants often correspond in part or whole to the reciprocals of the steady state coefficients. This is the major reason for using the formulation... 

In sharp contrast to the reductive half-reaction, where the free oxidized flavin is totally inert in the presence of physiological substrates, reduced model flavins are appreciably reactive (nonenzymatically) with O2 and other electron acceptors. However, the O2 reactivity of reduced flavin is complicated for two perhaps related reasons (61). First, the reaction is autocatalytic owing to the formation of 2F (from F and FH2) which in its anionic state is extremely reactive with Oo. Second, the superoxide radical is an important kinetic intermediate in O2 reduction (59). Neither of these features is observed with the reduced flavoprotein oxidases. 

DAAO is one of the most extensively studied flavoprotein oxidases. The homodimeric enzyme catalyzes the strictly stere-ospecihc oxidative deamination of neutral and hydrophobic D-amino acids to give a-keto acids and ammonia (Fig. 3a). In the reductive half-reaction the D-amino acid substrate is converted to the imino acid product via hydride transfer (21). During the oxidative half-reaction, the imino acid is released and hydrolyzed. Mammalian and yeast DAAO share the same catalytic mechanism, but they differ in kinetic mechanism, catalytic efficiency, substrate specificity, and protein stability. The dimeric structures of the mammalian enzymes show a head-to-head mode of monomer-monomer interaction, which is different from the head-to-tail mode of dimerization observed in Rhodotorula gracilis DAAO (20). Benzoate is a potent competitive inhibitor of mammalian DAAO. Binding of this ligand strengthens the apoenzyme-flavin interaction and increases the conformational stability of the porcine enzyme. 

Figure 4 Ternary complex kinetic mechanism of flavoprotein oxidases.

Recently, L-amino acid deaminase (EC 1.4.3.x) activities have been identified, particularly from the Proteus genus [59]. This enzyme, constituted by 370 residues, is an FAD-containing L-amino acid oxidase flavoprotein that uses molecular oxygen to convert L-amino acids into the corresponding a-keto adds and ammonia but does not produce hydrogen peroxide. L-amino acid deaminase prefers amino acids with aliphatic, aromatic, and sulfur-containing side chains (the best substrates are L-heu, L-Phe, L-Met, and L-Trp) and its kinetic efficiency is quite low because of the low Vnm value (<2 units/mg protein). 

A theoretical treatment of the kinetics of coenzyme-linked reactions by Stadie and Zapp revealed that D-amino acid oxidase has practically an identical affinity for the substrate (o-alanine) when the enzyme is in either the Zwitter or anion forms, the respective Km values being 8.7 X 10 and 9.2 X 10 M. Other estimates of K, are 5, and 6.1-6.6 X 10 M. The A tiavin-protem valuc is of the order of 2.5 X 10 M. Stadie and Zapp found that the flavoprotein may be treated as an acid with a pKof 7.9, so at pH 7.9 and 9 X 10 M alanine, the enzyme is half dissociated as an acid and both forms half combined with the substrate. 

So far, examples to illustrate experimental methods for following the time course of the approach to steady states and of their kinetic interpretation have been restricted to enzymes which do not have a natural chromophore attached to the protein although reference has been made to the classic studies of Chance with peroxidase (see p. 142). Qearly the application of these techniques to the study of enzymes with built in chromophores, such as the prosthetic groups riboflavine, pyridoxal phosphate or haem, contributed considerably to the elucidation of reaction mechanisms. However, the progress in the identification of the number and character of intermediates depended more on the improvements of spectral resolution of stopped-flow equipment than on any kinetic principles additional to those enunciated above. This is illustrated, for instance, by the progress made between the first transient kinetic study of the flavoprotein xanthine oxidase by Gutfreund Sturtevant (1959) and the much more detailed spectral analysis of intermediates by Olson et al. (1974) and Porras, Olson Palmer (1981). 

Walaas, E., and O. Walaas Kinetics and Equilibria in Flavoprotein Systems. V. The Effects of pFI, Anions and Partial Structural Analogues of the Coenzyme on the Activity of D-Amino Acid Oxidase. Acta Chem. Scand. 10, 122 (1956). 

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