US EPA

6251184 

Journal Article 

Steady-state kinetics of the tungsten containing aldehyde: ferredoxin oxidoreductases from the hyperthermophilic archaeon Pyrococcus furiosus 

Hagedoorn, PL 

2019 

Yes 

Journal of Biotechnology
ISSN: 0168-1656
EISSN: 1873-4863 

306 

142-148 

English 

31589889 

10.1016/j.jbiotec.2019.10.005 

WOS:000496193800018 

https://www.proquest.com/scholarly-journals/steady-state-kinetics-tungsten-containing/docview/2327996234/se-2?accountid=171501
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The tungsten containing Aldehyde:ferredoxin oxidoreductases (AOR) offer interesting opportunities for biocatalytic approaches towards aldehyde oxidation and carboxylic acid reduction. The hyperthermophilic archaeon Pyrococcus furiosus encodes five different AOR family members: glyceraldehyde-3-phosphate oxidoreductase (GAPOR), aldehyde oxidoreductase (AOR), and formaldehyde oxidoreductase (FOR), WOR4 and WOR5. GAPOR functions as a glycolytic enzyme and is highly specific for the substrate glyceraldehyde-3-phosphate (GAP). AOR, FOR and WOR5 have a broad substrate spectrum, and for WOR4 no substrate has been identified to date. As ambiguous kinetic parameters have been reported for different AOR family enzymes the steady state kinetics under different physiologically relevant conditions was explored. The GAPOR substrate GAP was found to degrade at 60 °C by non-enzymatic elimination of the phosphate group to methylglyoxal with a half-life t1/2 = 6.5 min. Methylglyoxal is not a substrate or inhibitor of GAPOR. D-GAP was identified as the only substrate oxidized by GAPOR, and the kinetics of the enzyme was unaffected by the presence of L-GAP, which makes GAPOR the first enantioselective enzyme of the AOR family. The steady-state kinetics of GAPOR showed partial substrate inhibition, which assumes the GAP inhibited form of the enzyme retains some activity. This inhibition was found to be alleviated completely by a 1 M NaCl resulting in increased enzyme activity at high substrate concentrations. GAPOR activity was strongly pH dependent, with the optimum at pH 9. At pH 9, the substrate is a divalent anion and, therefore, positively charged amino acid residues are likely to be involved in the binding of the substrate. FOR exhibited a significant primary kinetic isotope effect of the apparent Vmax for the deuterated substrate, formaldehyde-d2, which shows that the rate-determining step involves a CH bond break from the aldehyde. The implications of these results for the reaction mechanism of tungsten-containing AORs, are discussed. 

article; Tungsten enzyme; Glyceraldehyde-3-phosphate oxidoreductase; Aldehyde ferredoxin oxidoreductase; Steady state kinetics; Pyrococcus furiosus; amino acids; carboxylic acids; catalytic activity; enantioselectivity; enzyme activity; formaldehyde; glyceraldehyde 3-phosphate; glycolysis; half life; isotopes; oxidation; oxidoreductases; phosphates; reaction mechanisms; sodium chloride; tungsten