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• Glucose oxidase method: This couples the reaction to peroxidase and measures
  the absorption of the oxidised dye in the visible region or uses an oxygen electrode
  to measure the oxygen consumption directly:

D-glucoseþH 2 OþO 2 ÐD-gluconic acidþH 2 O 2

H 2 O 2 þdyereducedÐH 2 Oþdyeoxidised

The glucose oxidase method usesb-D-glucose as substrate but blood glucose contains

an equilibrium mixture of it and thea-isomer. Fortunately, preparations of glucose

oxidase contain an isomerase that interconverts the two isomers thus allowing the assay

of totalD-glucose. Examples of the commonly used dye are 4-aminophenazine and

o-dianisidine.

• Glucose dehydrogenase method: This requires no coupled reaction, but simply
  measures the increase in absorption at 340 nm:

b-D-glucoseþNADþÐD-glucono-1;5-lactoneþNADHþHþ

Another blood substrate commonly assayed by enzyme-based techniques in clinical

biochemistry is cholesterol:

• Cholesterol: This is used as an indicator of atherosclerosis and susceptibility to coronary
  heart disease (Section 18.2.2). It uses cholesterol oxidase and peroxidase and measures
  the absorption in the visible region due to the oxidised dye, for example 4-amino-
  phenazine:

cholesterolþO 2 Ð4-cholesten-3-oneþH 2 O 2

H 2 O 2 þdyereducedÐH 2 Oþdyeoxidised

Any cholesterol ester in the sample is hydrolysed to free cholesterol by the inclusion

of cholesterol esterase in the reaction mixture.

Biosensors (Sections 1.3.5 and 16.2.2) such as those for glucose and cholesterol are

based on the above reactions and afford a simple means for the fast measurement of

these substrates.

15.4 Enzyme active sites and catalytic mechanisms

15.4.1 Enzyme active sites

As previously pointed out, enzymes are characterised by their high specificity, cata-

lytic activity and capacity for regulation. These properties must reflect the specific

three-dimensional interaction between the enzyme and its substrate. A complete

understanding of the way enzymes work must therefore include the elucidation of

611 15.4 Enzyme active sites and catalytic mechanisms