KEY ROLE OF ALKANOIC ACIDS ON THE SPECTRAL PROPERTIES, ACTIVITY, AND ACTIVE-SITE STABILITY OF IRON-CONTAINING NITRILE HYDRATASE FROM BREVIBACTERIUM-R312

Interaction of n-butyric acid with dialyzed nitrile hydratase from Bre vibacterium R312, which is characterized by a charge-transfer band at 680 nm and EPR signals typical of a low-spin Fe(III) with Delta g = 0. 22, leads to a form displaying different spectral properties (lambda = 710 nm, Delta g = 0.31). Butyric acid also acts as a competitive inhi bitor of nitrile-hydratase-catalyzed hydration of acrylonitrile with a K-i value of 0.9 mM. Formation of the complex between the enzyme and butyric acid is highly dependent on the concentration of the latter an d on pH. When stored with high levels of butyric acid, nitrile hydrata se is completely inactive. The active uncomplexed enzyme is restored u nder the high dilution conditions used for the enzymatic assays, while the complexed form is favored at acidic pH and is not formed at pH ab ove 8. Furthermore, the inhibitory potency of butyric acid decreases u pon increasing pH (IC50 increases from 0.8 mM at pH 6.2 to 12 mM at pH 8.2). These data show that nitrile hydratase interacts with the acid form of butyric acid with a high affinty (K-i' approximate to 4 mu M a t pH 7.2). At pH <3, the visible spectrum of the enzyme disappears, pr esumably because of demetallation, whereas that of the complex exhibit s a charge-transfer band shifted to 800 nm, the presence of butyric ac id preventing nitrile hydratase from demet allation. Other linear carb oxylic acids such as valeric and hexanoic acids behave similarly; they act as inhibitors of nitrile hydratase and protect the enzyme during storage. A structure of the nitrile hydratase active site interacting with butyric acid is tentatively proposed in which the latter is hydro gen-bonded to the Fe(III)-OH moiety. This interaction between butyric acid and nitrile hydratase should be considered when deducing the natu re of nitrile hydratase active site and mechanism, from spectral and e nzymatic data, since most results published previously have been obtai ned on nitrile hydratase containing large amounts of butyric acid and interpreted without taking into account the presence of this acid in t he active site.