F. Effects of pH, Temperature, Ionic Strength, and Solvent

Figure 7 gives a typical behavior of an enzyme as a function of pH. The apparent pH optimum for the activity of an enzyme can be due to reversible effects on the velocity, an effect on the affinity of a substrate, or an effect on enzyme stability [33,34]. These can be distinguished experimentally. Stability can be measured by preincubation of the enzyme at different pHs before assaying activity after standardizing the pH to that in the assay. This effect would be time dependent and nonreversible and can be eliminated by short incubation times. A reversible ionization that affects catalytic groups but does not affect enzyme stability would give the same activity after readjustment to the optimal pH.

Effects of pH on substrate affinity can be eliminated by using a sufficiently high concentration of substrate to saturate the enzyme at all pHs used. But this does require a check of the Km as a function of pH to ensure that at the assay pH the substrate is still several fold Km. At high [S], all enzyme is complexed as ES, so changes in the state of ionization of the free enzyme or free S will not affect Vmax, only Km.

Figure 7 pH dependence of enzyme activity with acidic and basic titratable groups affecting velocity.

The pH effects on velocity [54] are more complex and depend upon the ionizing groups, their proximity to the active center, and the breakdown path and rates of each ionized species. Velocity measurements at saturating substrate (Vmax conditions) yield pKas of the ES forms. Velocity measurements at subsaturating substrate (Vmax/Km conditions) yield pKas of both free E and S.

Inhibition of enzymes may be greatly affected by pH in both the reversible and irreversible case if an ionizable group is involved in the binding or inactiva-tion. The pH chosen for a screen can also have direct effects on the apparent inhibitory potency of compounds being screened. In the case of stromelysin, the inhibitory potencies of peptide phosphonamidates were highest at lower pH (pH 6), so a screen run at the typical ''physiological'' pH 7.4 would have missed this class of compound [35].

Temperature can affect the stability of the enzyme, Vmax, Km, or the pKa of functional groups. It can also affect activators or different enzymes in the assay. While this might suggest that the effects of temperature are extremely complex, actually they can be readily distinguished experimentally. Stability effects can be studied by preincubation at temperatures prior to assaying. Effects

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