Studies of NADP<sup>+</sup>-preferred secondary alcohol dehydrogenase from <i>Thermoanaerobium brockii</i>

Al-Kassim, Loola; Tsai, Chin‐Chun

doi:10.1139/o90-135

Cited by 19 publications

(9 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(2)] where KA, KB, and KIA are the Michaelis constant for substrate A, the Michaelis constant for substrate B, and the dissociation constant for substrate A, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…(2)], and the corresponding double reciprocal plots intersect to the left of the vertical axis (Fig. 1).…”

Section: Substrate Concentration and Haldane Relationshipmentioning

confidence: 91%

“…(2). The parameter for the other substrates are from experiments in which the alcohol, aldehyde, or the ketone was the variable substrate in the presence of a constant, saturating concentration of nucleotide.…”

Section: Substrate Specificitymentioning

confidence: 99%

See 2 more Smart Citations

Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

Jb¹,

Kj²,

Kb³

1993

Biotech & Bioengineering

View full text Add to dashboard Cite

Alcohol dehydrogenase (E. C. 1.1.1.1) from Thermoanaerobium brockii at 25 degrees C and at 65 degrees C is more active with secondary than primary alcohols. The enzyme utilizes NADP and NADPH as cosubstrates better than NAD and NADH. The maximum velocities (V(m)) for secondary alcohols at 65 degrees C are 10 to 100 times higher than those at 25 degrees C, whereas the K(m) values are more comparable.At both 25 degrees C and 65 degrees C the substrate analogue 1,1,1,3,3,3-hexafluoro-2-propanol inhibited the oxidation of alcohol competitively with respect to cyclopentanol, and uncompetitively with respect to NADP. Dimethylsulfoxide inhibited the reduction of cyclopentanone competitively with respect to cyclopentanone, and uncompetitively with respect to NADPH. As a product inhibitor, NADP was competitive with respect to NADPH. These results demonstrate that the enzyme binds the nucleotide and then the alcohol or ketone to form a ternary complex which is converted to a product ternary complex that releases product and nucleotide in that order.At 25 degrees C, all aldehydes and ketones examined inhibited the enzyme at concentrations above their Michaelis constants. The substrate inhibition by cyclopentanone was incomplete, and it was uncompetitive with respect to NADPH. Furthermore, cyclopentanone as a product inhibitor showed intercept-linear, slope-parabolic inhibition with respect to cyclopentanol. These results indicate that cyclopentanone binds to the enzyme-NADP complex at high concentrations. The resulting ternary complex slowly dissociates NADP and cyclopentanone.At 65 degrees C, all of the secondary alcohols, with the exception of cyclohexanol, show substrate activation at high concentration. Experiments in which NADP was the variable substrate and cyclopentanol as the constant-variable substrate over a wide range of concentrations gave double reciprocal plots in which the intercepts showed substrate activation and the slopes showed substrate inhibition. These results indicate that the secondary alcohols bind to the enzyme-NADPH complex at high concentrations and that the resulting ternary complex dissociates NADPH faster than the enzyme-NADPH complex.

show abstract

“…(2)] where KA, KB, and KIA are the Michaelis constant for substrate A, the Michaelis constant for substrate B, and the dissociation constant for substrate A, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…(2)], and the corresponding double reciprocal plots intersect to the left of the vertical axis (Fig. 1).…”

Section: Substrate Concentration and Haldane Relationshipmentioning

confidence: 91%

See 1 more Smart Citation

Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

Jb¹,

Kj²,

Kb³

1993

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…Initial velocities were fitted with Equation 1 (as described in al‐Kassim et al 1990) by nonlinear regression using the Solver tool implemented in Microsoft Excel: …”

Section: Methodsmentioning

confidence: 99%

The conserved Glu‐60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

et al. 2003

View full text Add to dashboard Cite

Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four-and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover.Keywords: Alcohol dehydrogenase; metalloenzyme; glutamate; active site; X-ray absorption Interconversions of alcohols, aldehydes, and ketones are essential processes in both prokaryotes and eukaryotes. Several catalytic mechanisms for alcohol dehydrogenases (ADHs) have been proposed based on accumulating structural and spectroscopic evidence gathered from the moststudied enzyme, horse liver ADH (HLADH; Bertini 1986; Wilkinson 1987). The oxidation of alcohols requires a net removal of two hydrogen atoms from the substrate. This dehydrogenation process is known to proceed by coupled processes of proton abstraction and hydride ion transfer. The two main classes of structural mechanisms based on a proton relay system that have been proposed for HLADH differ specifically in the hypothesized coordination of the zinc ion during catalysis. One mechanism involves the displacement of the zinc-bound water by the alcohol substrate, and so, the zinc ion remains tetrahedrally coordinated during catalysis (Dunn et al. 1975;Eklund et al. 1982). Alternatively, the substrate molecule is added to the tetrahedral zinc ion to form penta-coordinated zinc (Dworschack and Plapp 1977;Makinen et al. 1983).Thermoanaerobacter brockii ADH (TbADH) is a medium-chain, NADP + -linked (Lamed and Zeikus 1981) class-A enzyme (Peretz et al. 1993) that reversibly catalyzes the oxidation of secondary alcohols to the corresponding ketones. TbADH is a tetramer comprising four identical Abbreviations: ADH, alcohol dehydrogenase; CbADH, Clostridium beijerinckii alcohol dehydrogenase; TbADH, Thermoanaerobacter brockii alcohol dehydrogenase; EXAFS, extended X-ra...

show abstract

“…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.…”

Section: Generalmentioning

confidence: 99%

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

McMahon

Mulcahy

2002

Biotech & Bioengineering

View full text Add to dashboard Cite

This study describes the development and application of a bioaffinity chromatographic system for the one-step purification of an NADP(+)-dependent secondary alcohol dehydrogenase from the obligate anaerobe, Thermoanaerobacter brockii (TBADH, EC 1.1.1.2). The general approach is based upon improving the selectivity of immobilized cofactor derivatives (general ligand approach to bioaffinity chromatography) through using soluble enzyme-specific substrate analogues in irrigants to promote biospecific adsorption (the kinetic locking-on tactic). Specifically, the following is described: Evaluation of 8'-azo-linked, C(8)-linked, N(1)-linked, and N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of TBADH; evaluation of 2', 5'-ADP as a stripping ligand for TBADH bioaffinity purifications using an 8'-azo-linked immobilized NADP(+) derivative in the locking-on mode; and application of the developed bioaffinity chromatographic system to the purification of TBADH from a crude cellular extract. Surprizingly, of the four immobilized NADP(+) derivatives investigated, only the 8'-azo-linked immobilized NADP(+) derivative proved effective for TBADH affinity purification when used in conjunction with pyrazole (a competitive inhibitor of TBADH activity) as the locking-on ligand. This is in contrast to other NADP(+)-dependent dehydrogenases where the immobilized N(6)-linked cofactor proved to be suitable. While the one-step purification of TBADH to electrophoretic homogeneity is described in the present study (92% yield), results from the model chromatographic studies point to improvements that could be made to the immobilized cofactor derivative to improve its suitability for TBADH bioaffinity purification and to facilitate future large scale protein purification operations.

show abstract

Studies of NADP⁺-preferred secondary alcohol dehydrogenase from Thermoanaerobium brockii

Cited by 19 publications

References 27 publications

Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

The conserved Glu‐60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

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

Contact Info

Product

Resources

About

Studies of NADP+-preferred secondary alcohol dehydrogenase from Thermoanaerobium brockii

Cited by 19 publications

References 27 publications

Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

The conserved Glu‐60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

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

Contact Info

Product

Resources

About

Studies of NADP⁺-preferred secondary alcohol dehydrogenase from Thermoanaerobium brockii

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