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• Isotope exchange studies:The replacement of the natural isotope of an atom in the
  substrate by a different isotope of the same element and the study of the impact of
  the isotope replacement on the observed rate of enzymatic reaction and its associated
  stereoselectivity, often enables deductions to be made about the mechanism of the
  reaction. Two examples illustrate the principle. Firstly, alcohol dehydrogenase (AD)
  that oxidises ethanol to ethanal using NADþ:NADH:

CH 3 CH 2 OHþNADþ^ !

AD

CH 3 CHOþNADHþH

ethanol ethanal

The two hydrogen atoms on the methylene (CH 2 ) group of ethanol are chemically

indistinguishable, but if one is replaced by a deuterium or tritium atom the carbon

atom becomes a chiral centre and the resulting molecule can be identified as eitherR

orSconfiguration according to the Cahn–Ingold–Prelog rule for defining the stereo-

chemistry of asymmetric centres. Studies have shown that alcohol dehydrogenase

exclusively removes the hydrogen atom in the proRconfiguration, i.e. (R)CH 3 CHDOH

always loses the D isotope in its conversion to ethanal but (S)CH 3 CHDOH retains it.

Such a finding can only be interpreted in terms of the specific orientation of the

ethanol molecule at the binding site such that the two hydrogen atoms are effectively not

equivalent. All dehydrogenases have been shown to display this type of stereospecificity

and can be classified as either A-side dehydrogenases (e.g. alcohol dehydrogenase,

lactate dehydrogenase, malate dehydrogenase) or B-side dehydrogenases (e.g. glycerol

3-phosphate dehydrogenase, glucose dehydrogenase, glyceraldehyde-3-phosphate

dehydrogenase). Interestingly, the class type is independent of the hydrogen acceptor

being NADþor NADPþ. Secondly, the hydrolysis of esters by esterases that convert the

ester to a mixture of acid and alcohol simultaneously incorporating a molecule of water

into the products:

RCOOR^0 þ H 2 O !esterase RCOOH þ R^0 OH

ester acid alcohol

Inthis reaction the oxygen atom identified as O* caneitherbe retained inthe acid orin the

alcohol depending upon which side of the labelled oxygen atom the bond is broken, with

water providing the second oxygen atom in the products. Labelling the oxygen atom in

question as^18 O and studying, by mass spectrometry, its location after hydrolysis enables

details to be drawn about the mechanism of the hydrolysis of the ester by the esterase.

In practice, the labelled oxygen is found in the alcohol which supports the view that the

reaction mechanism involves initial attack by water, acting as a nucleophile, on the

carbonyl carbon atom and the subsequent elimination of the R^0 O* group.

• Site-directed mutagenesis studies:Advances in molecular biology and particularly
  in the ability to clone genes and express them in a particular vector have opened up
  the possibility of producing variants of the enzyme in which a particular amino acid
  residue, thought to be involved in substrate binding and catalysis, is replaced by
  another amino acid. By studying the impact of the replacement of an ionisable or

613 15.4 Enzyme active sites and catalytic mechanisms