Ethanol is a good substrate for YADH. It is known to react with
many types of alcohol.11 YADH-1 prefers to catalyse acetaldehyde to
ethanol due to its low Km= 53.12 mM-1, indicative of high affinity
for YADH (fig. 5b), requiring less substrate to reach ½ Vmax. Vmax=
0.065 µmol/min-1. Therefore, ideal for YADH and has high efficiency
for product formation. It is readily made to orient the transfer of hydride from
The long chains, steric hindrance result in high Km as
in propan-1-ol and propan-2-ol. Propan-1-ol, Km= 60 mM-1;
Vmax= 0.021 µmol/min-1 is increasingly less effective. The
substrate binding is deemed to be small in Saccharomyces cerevisiae
compare to those in liver, due to Trp-58, Trp-57 and Thr-48.11
This further test a proposal made that low rates from large alcohols are due to
the size of substrate pocket. Propan-2-ol, Vmax= 6.55×104
µmol/min-1; Km= 67 mM-1. Secondary alcohol, increasingly
less effective (fig. 6b) as substrate than ethanol and propan-1-ol. It
structurally has OH bonded two carbons away; with a bulky ‘Y’ shape preventing
the binding of ethanol to YADH.10
2,2,2 trifluoroethanol is a competitive inhibitor and unreactive,
dissociating constant, Ki= 4.1 mM; TFE= -5×10-3 mM. Has
a similar structure to ethanol (primary structure), which gives it characteristic
to be competitive at the active site. It binds tightly to enzyme-NAD+
compound. Affecting the transfer of hydride ion from NAD+ ? 1o
Carbon due to high electronegative fluorine charge.
His and Tyr become affected at the active site changes
conformation as the reaction moves towards and beyond the optimum, pH 9 (fig.
7a). the Km values of pH increases as pH moves away from the optimum
(fig. 8b). This is due to the charges on the active site. In extreme pH,
chemical bonds in YADH, holding the specific tertiary structure (fig. 1) are
broken, causing denaturation of YADH.
In conclusion, YADH works efficiently with small alcohol as
substrates, but affected by factors such as inhibitors and pH. Causing the Vmax
and Km to decrease. For further research, different types of
inhibitors can be investigated as well as tertiary alcohols.