Kinetics of lmidazole Catalyzed Ester Hydrolysis Use of Buffer Dilutions to Determine Spontaneous Rate, Catalyzed Rate, and Reaction Order Anthony Lombardo Florida Atlantic University, Boca Raton, FL 33431 Described herein is an advanced undergraduate kinetics experiment which yields a great deal of information from an easily obtained data set of modest size. It is designed to extract the individual rate constants for a reaction proceeding by a catalyzed and uncatalyzed pathway simultaneously. This situation is not uncommon in aqueous solution where weak bases introduced as buffers can sometimes act as catalytic species as well. The reaction utilized is hydrolysis of p-nitrophenyl acetate (NPA) in the presence of imidazole (Im) which has been shown to enhance the rate of the reaction (I).This system 0
nueleophilie catalysis
In either mechanism, the buffer species, Im, is a direct participant in the transition state. Thus, i t appears in the rate law which is written as shown in eqn. (1).The first-order rate constant, ko is rate = (ko+ k,t[Im]n)[NPA]
was first employed as a model t o investigate participation of the histidine side chain in the catalytic functions of hydrolytic enzymes such as trypsin, chymotrypsin, and acetylcholinesterase (2,3).Thus, this experiment is also a useful tool in teaching fundamental mechanistic principles operative in reactions of biochemical sienificance. The catalyzed hydrolysi;' ran proceed by two distinct but kinetirallv indi~tincuishableimthwavs: (1) general base catalysis in"which lm enhances the n;cliop6ilicity of water through partial proton donation, and (2) nucleophilic catalysis in which N-acetylimidazole is an intermediate of considerable stability. These are shown below. ~
~~~~
~
~
~
(1)
for the uncatalvzed hvdrolvsis (oarticioation of solvent soecies only), and kCat-ist h e b e c o ~ h o r d eraie r constant for catalysis by buffer. The aim of this experiment is to evaluate the extent of rate enhancement due to Im by determining ko and hat, and to confirm that the catalyzed process is first-order in Im as required by either mechanism.' This is done by buffer dilutions. Under pseudo first-order conditions in which [Im] >> [NPA], eqn. (1) reduces to (2) from which eqn. (3) follows. Note that rate = k,b[NPA]
(2)
eqn. (3) has the form of a straight line if the reaction is firstorder in Im. Thus, kinetic runs are performed in which pH and [NPA] are held constant while [Im] is varied from run to run (i.e., a buffer dilution experiment). Then, the resuitant pseudo first-order k,b values (see Experimental Procedure) are plotted ngains;their ~ o r r e s p o n d l n[lm]. ~ Alinear rurveconfirms first-order kinetics in lm. The slope of the line will he kat and its intercept (extrapolation of line t o zero buffer concentration) is ko. In the following section, a series of buffer dilutions done a t a single pH is described. This experiment can be enhanced by running dilutions a t a number of pH values. In this manner the catalytic species of Im is revealed t o he the free base (as the mechanisms require), and the pH dependence of ko can be investigated to determine the participating solvent species (4).
Experimental Procedures Mnterialr. 1m and N P A can he purchased from any uf a number
