Was the Appropriate Range Used

Obtaining accurate measurements of Km is important because Km provides a quantitative measure of enzyme-substrate complementarity in binding (when Km Ks), and such values can be used to compare relative affinities of competing substrates. Second, the combined determination of Vmax (oc cat) and Km for competing substrates provides for a quantitative comparison of specificity (selectivity) of the enzyme among substrates through the use of the specificity constant, or VmaxZ-K m [Eq. (14.4)] (Fersht, 1985). 

Studies that seek to compare specificity constants among different substrates under a defined set of conditions are often focused on the nature of enzyme-substrate interaction or structure-function relationships that confer reaction selectivity. In other cases, the determination of specificity constants for a single substrate under a variety of conditions is often an attempt to infer something about factors that govern or modulate reaction selectivity. In both cases, obtaining reliable data and estimates of kinetic constants are of paramount importance. The collection of observations in Table 14.1 provides an example of such a study, where different substrates were assayed over different ranges of [S] at a known [E] to yield estimates of fccat and Km- 

The conclusions to be drawn for this type of study are likely to focus on the relationship between systematic changes in stmctural features of the substrates and the attendant changes in reaction selectivity (relative kcailKm values). This may allow certain inferences to be drawn about the chemical nature of enzyme-substrate interactions that lead to productive binding and/or transition-state stabilization. 

For example, a possible conclusion to be reached from the data in Table 14.1 is Reaction selectivity with substrate 7 was two orders of magnitude greater than for substrates 5 or 6 . Based on structural differences between substrate 7, and 5 and 6, conclusions may be further delineated to suggest that specific functional groups of the substrate (and enzyme) may participate in catalysis by facilitating substrate binding or substrate transformation. Such conclusions would be vaUd or at least firmly supported if measurements of cat and Km are accurate and reliable (Table 14.1). 

It is a rather simple task to judge the reliability of this data set by calculating the Km value (from the fourth and fifth colunms in Table 14.1) and comparing it to the range of [S] values used (the second colunm in 

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