[6 Active Site Titration of Peptidases

By C. Graham Knight

Introduction

Measurement of the active-site molarity of a peptidase is a valuable step toward defining the molecular properties of the enzyme. In the absence of such a method, catalytic activity (micromoles of substrate cleaved per second) can only be defined in terms of protein concentration. Protein concentration may be measured directly or derived from an established assay for which a specific activity was calculated on the basis that the purified protein was fully active. This assumption is likely to be erroneous, for even with rapid and mild methods of protein isolation, a substantial proportion of the enzyme protein in a purified preparation may be inactive, perhaps as a result of modifications in vivo prior to isolation. Although affinity purification may overcome this objection, enzymes vary widely in lability, and a fully active preparation cannot be assumed. If only small quantities are available, the concentration may have been determined by comparison to a standard protein such as albumin which can cause further errors. Although measurements of absorbance can be related to the residual weight of exhaustively dried solutions, tightly bound water or nonvolatile impurities will lead to error.

In studies with recombinant enzymes, it is essential to measure active-site concentrations, so that the effect of amino acid substitutions can be correctly ascribed to effects on catalysis rather than effects on the stability of the protein. Active-site titration, by placing the enzyme activity on a molar basis, also allows the absolute comparison of kinetic constants obtained on different occasions and in different laboratories. In contrast, comparisons of activity are likely to be influenced by differences in the assay conditions or the presence of inhibitory impurities.

The criteria which a titrant should fulfill remain those defined for the first reagents of this type.1 The titrant should be a stable, well-characterized molecule that combines rapidly only with the active site with 1:1 stoichiom-etry to yield either an inactive or a slowly turned-over complex. Three classes of reagents are potential titrants: substrates that form a stable intermediate acyl-enzyme with the release of an easily quantified first product,

1 M. L. Bender, M. L. Begue-Canton, R. L. Blakely, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. Kezdy, J. V. Killheffer, Jr., T. H. Marshall, C. G. Miller, R. W. Roeske, and J. K. Stoops, J. Am. Chem. Soc. 88, 5890 (1966).

METHODS IN ENZYMOLOGY, VOL. 248

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

irreversible inhibitors that react with a single active-site residue, and tight-binding reversible inhibitors. Each of the classes will be considered in turn and representative examples presented.

Burst Titrations with Specific Substrates Kinetic Analysis

The first titrants were developed for serine and cysteine proteinases following the discovery of the acyl-enzyme mechanism.2 The kinetics of substrate turnover by such enzymes is described by Eq. (1), where Ks is

+ P, the dissociation constant of the initial noncovalent E • S complex, k2 is the first-order acylation rate constant, and k3 the deacylation constant. If k3 < k2, there will be an initial rapid release, or "burst," of the first product Pi on mixing enzyme and substrate. This accompanies the buildup of the steady-state concentration of the acyl-enzyme, ES', and is followed by a slower rate of turnover. A classic example of biphasic behavior is the burst of p-nitrophenol released during the reaction of trypsin with a large molar excess of p-nitrophenyl guanidinobenzoate.3 When [S]Q §> [E]0, the final steady-state portion of the curve can be described by

where tt is the initial burst of 4-nitrophenol and v is the final steady-state velocity. The magnitude of tt is given by w = [E]0 {k2/(k2 + A:3)}2/(1 + *m/[S]0)2 (3)

Inspection of Eq. (3) reveals that tt = [E]0 only when k2 > k3 and [S]0 8> Km, where §> means "at least 100 times greater than." When these conditions are met, most commonly by using chemically reactive phenolic esters as substrates, the size of the burst is independent of titrant concentration. If the condition that [S]Q > Km cannot be met, the burst should be measured over a range of titrant concentrations at a constant value of [E]0. The data are plotted as 1 /tt112 versus 1 /[S]Q, and the intercept on the ordinate gives (k2 + k3)/(k2 [E]'/2). It must be emphasized that there is no comparable procedure to derive [E]0 if k3 ~ k2 and for this reason burst titrations

2 B. S. Hartley and B. A. Kilby, Biochem. J. 56, 288 (1954).

3 T. Chase, Jr., and E. Shaw, this series, Vol. 19, p. 20.

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