Use of steady-state techniques

Steady-state techniques remain a major mode of enzyme investigation forming the basis of most enzyme assays, inhibition studies, pH profiles, etc. The detailed kinetics of this area are discussed well elsewhere in this volume [1] and our coverage here is selective of the common uses of steady-state kinetics. 

In spite of their structural complexity and, indeed of the often chemically highly complicated processes which they catalyse, enzymes acting in the steady-stale frequently follow a very simple hyperboUc rate equation, the Michaelis-Menten equation (Eqn. 6), where Eg. 

Sq AT ,). Eq and Sq are the initial enzyme and substrate concentrations respectively and is an operationally defined coefficient, the Michaelis constant, which simply provides the substrate concentration required to attain half-maximal velocity 

For the purposes of our discussion we shall look in turn at the uses of each term in Eqn. 6. The Cq term should be noted to be a velocity (units are concentration or amount/time) and not a rate coefficient. The terms and K (whose components can sometimes be dissected by means of fast reaction techniques, vide infra) are used to describe the sensitivity of the enzyme reaction to a variety of changes, as described above. Their ratio k yK, with dimensions of a second-order rate coefficient (M sec ) is also useful and has been called the specificity constant by Brot and Bender [4]. For a single, covalent intermediate pathway (e.g. Eqn. 7, e.g. an acyl-enzyme pathway, this composite constant is insensitive to problems such as non-productive substrate binding, whilst its components are complicated by such problems. 

It is clear that whatever the relative values of Sq and in Eqn. 6, as long as o observed initial velocity is proportional to the concentration of functioning enzyme (Eq) present in solution. This is the basis of rate assays for enzymes. By working at Sq K, i.e. Vq = Pj ax the observed velocity is not complicated by errors made in values of Sq. To obtain a value of Eq itself from such a velocity measurement requires at least an accurate knowledge of (for Sq /Tm 

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