Kinetic mechanism of the oxidation of 2-propanol catalyzed by Thermoanaerobium brockii alcohol dehydrogenase

A Kinetic Locking-On Strategy for Bioaffinity Purification: Further Studies with Alcohol Dehydrogenase

“…One example of this was observed during affinity chromatographic studies on TBADH. Kinetic experiments on TBADH have shown that pyrazole competitively inhibits 2-propanol oxidation (15). Furthermore, although hydroxylamine has been shown to be a competitive inhibitor of both yeast and horse liver enzymes (16), its inhibitory effect on TBADH has not been reported.…”

“…Since this enzyme is an NADP ϩ -dependent dehydrogenase, all of the locking-on studies were performed on 8Ј-azo-linked and N 6 -linked immobilized NADP ϩ derivatives. This enzyme is competitively inhibited by both pyrazole and hydroxylamine (with K i values of 0.2 and 0.4 mM, see Table 2) and it is believed to follow a sequential ordered mechanism of substrate binding and product release (15). It thus seemed a prime candidate for purification using the locking-on tactic.…”

New kinetic and thermodynamic approach of one-pot synthesis of chiral alcohol by oxidoreductase from Candida parapsilosis

“…Recent literature [24][25][26] showed that some of these enzymes will follow a special form of ordered BiBi mechanism, the TheorellChance BiBi mechanism, which does not form central ternary complexes. The simplest method, the study of product inhibition caused by the first product of the reduction reaction compared to the second substrate of the carbonyl reductase, was carried out to analyze the kinetic significance of the ternary complexes (Table 3).…”

Bioaffinity purification of NADP+‐dependent dehydrogenases: Studies with alcohol dehydrogenase from Thermoanaerobacter brockii

“…This approach exploits the kinetic mechanism of the target enzyme for bioanity puri®cation applications and is applicable to those enzymes having an ordered sequential mechanisms (McMahon et al, 2001;Mulcahy et al, 1997aMulcahy et al, , 1997bMulcahy et al, , 1999Oakey et al, 1999;OÕCarra et al, 1996;OÕFlaherty et al, 1999aOÕFlaherty et al, , 1999bOÕFlaherty et al, , 1999cTynan et al, 2000). Because one of the primary potential advantages of this approach is that the same bioanity system should be applicable to the same/similar enzyme activity from very dierent sources (because separation is based on biospeci®city as opposed to gross physio- chemical properties), the present study aimed to apply the kinetic locking-on system designed for the eucaryotic NAD + -dependent ADH (YADH, EC 1.1.1.1), to bioanity puri®cation of an NADP + -dependent secondary ADH from the procaryotic obligate anaerobe, Thermoanaerobacter brockii (TBADH, EC 1.1.1.2; Al-Kassim and Tsai, 1990;Bogin et al, 1997;Oestreicher et al, 1996;Pereira et al, 1994). TBADH has been puri®ed using conventional chromatographic methods by several investigators.…”

“…These results with TBADH are in direct contrast to those obtained with other NADP + /NAD(P) + dependent dehydrogenases where an immobilized N 6 -linked cofactor proved eective for locking-on purposes (McMahon et al, 2001). Because TBADH is reported to exhibit a sequential ordered kinetic mechanism (Pereira et al, 1994), the most plausible explanation for failure to lock-on to the N 6 -linked immobilized NADP + was that the position of attachment of the cofactor impeded the binding of TBADH. In theory, this could have been established by attempting to enzymatically reduce the immobilized NADP + with TBADH in the presence of 2-propanol.…”