The kinetic mechanism of LDH is referenced, Ethanol can also undergo a very simi

Question

The kinetic mechanism of LDH is referenced,

Ethanol can also undergo a very similar reaction:

ethanol + NAD+ acetaldehyde + NADH

However, ethanol is a very poor substrate for LDH

Would you expect the Km for ethanol to be higher or lower than that for lactate?

Justify your answer with reference to the active site structure and the structures of lactate and ethanol.

Include sketches of the structures of lactate and ethanol at pH 7.0

(10 points)

2. We measured the kinetic parameters in the lactate to pyruvate direction, by following the formation of NADH from NAD+. The reaction can be measured in the reverse direction, but cannot be monitored at 340nm at high NADH concentrations – why is this? No more than two or three sentences, please (10 points)

Explanation / Answer

1.Km for LDH would be higher for ethanol than lactate as as Km is inverse to substrate affinity.

In LDH Arg109,Arg171,His195 are the catalytic sites that play an important role in catalysis.

Structure for ethanol= CH3CH2OH

Lacate=CH3CH(OH)COOH

The catalytic sites of LDH involves the use of nucleophilic/electrophilic reactions between the enzyme and the substrate and catalytic reaction takes place more efficiently for lactate as lactate orients itself better compared to ethanol in its active site.

2.Lactate+ NAD+=pyruvate+NADH+H+. Formation of NADH can be measure at 340 nm as nicotinamide moeity of NAD has high specific absorption when protonated (NADH form). Thus the measurement can be performed. High NADH means that spectrophotometer already gets saturated at 340 nm. Other wavelength might have to bee used other than 340 nm for the reverse reaction to be measured.

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