Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Enzymes
Determination of Enzyme Activity

Determining the quantitative content of Enzymes in biological objects presents certain difficulties because, with rare exceptions, enzymes are present in Tissues in negligible concentrations. Therefore, The amount of an enzyme is assessed by The rate of the catalyzed reaction under specific, standardized measurement conditions. Under optimal conditions of Temperature, medium pH, and complete enzyme saturation with the substrate, the rate of the catalyzed reaction is proportional to the Enzyme Concentration. The rate of an enzymatic reaction is judged either by the rate of substrate depletion or by the rate of product formation. To express enzyme concentration and quantitatively evaluate its activity in conventional units, the Enzyme Commission of the International Union of Biochemistry recommended the standard international unit (E or U): one unit of activity of any enzyme is defined as the amount that catalyzes The conversion of 1 micromole of substrate or The formation of 1 micromole of product per minute (μmol/min) under optimal conditions *.

In connection with the Introduction of the International System of Units (SI), a new expression for enzyme activity has been proposed in katals (kat, kat): 1 kat is the catalytic activity capable of carrying out a reaction at a rate of 1 mole per second (1 mol/s). The relation between the international unit (U) and the katal can be expressed as follows: 1 kat = 1 mol ∙ s^-1 = 60 mol ∙ min^-1 = 60 ∙ 10⁶ μmol ∙ min^-1 = 6 ∙ 10⁷ U, or: 1 U = 1 μmol ∙ min^-1 = (1/60) μmol ∙ s^-1 = (1/60) μkat = 16.67 nkat. Thus, 1 U of enzyme corresponds to 16.67 nkat.

It is also recommended to measure enzyme activity at 25°C, the optimum pH, and a Substrate Concentration exceeding saturation levels. Under these conditions, the reaction rate follows zero-order kinetics with respect to the substrate and depends solely on the enzyme concentration.

* Micromoles, 10^-6 moles; units of enzyme activity are also expressed in nanomoles (10^-9 moles) and picomoles (10^-12 moles).

In practical enzymology, specific and molar activities are frequently expressed using arbitrary conventions. Specific enzyme activity is typically defined as the number of units of enzymatic activity per milligram of protein (or katal units per kilogram of active protein). The number of substrate molecules converted into product per enzyme molecule per unit time under complete substrate saturation is referred to as the turnover number, or molar activity (molar catalytic activity is expressed in katals per gram-mole of enzyme). For instance, a single erythrocyte catalase molecule is capable of breaking down 44,000 hydrogen peroxide molecules per second*.