19 Rapid Reaction Kinetics Involving the Bioinorganic Chemistry Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 10/26/18. For personal use only.
Iron-Porphyrin Site of Horseradish Peroxidase H. B. DUNFORD and B. B. HASINOFF University of Alberta, Edmonton, Alberta, Canada
Kinetic studies of reactions of horseradish peroxidase (HRP) using stopped-flow and temperature-jump techniques are summarized. The reactions were studied intensively as a function of pH to establish the presence or absence of pH -dependences in the reaction rates. Minimum mechanisms are presented which cannot be proven to be correct. How ever, simpler mechanisms will not fit the data within experi mental error. The reactions which have been studied are fluoride and cyanide binding by (dissociation from) HRP and the oxidation of ferrocyanide to ferricyanide by HRP Compounds I and II. From the pH profiles of the reaction rates, the pK values of acid groups which influence the rates are deduced. Trends in p K values can be explained qualitatively in terms of electrostatic effects.
Τ Torseradish peroxidase has played an historical role in the development of enzyme chemistry. In the 1920s, Willstatter believed that the enzymatic activity of horseradish peroxidase ( H R P ) was caused by traces of inorganic material, a conclusion which he thought was applicable to other enzymes. H e thus remained skeptical of the results of Sumner, who obtained urease i n crystalline form and reported no inorganic elements present. H R P is now known to have a prosthetic group, a porphyrin ring system with a ferric iron at its center known as ferriprotoporphyrin I X or hemin (Figure 1). This is the same group found i n catalase, ferricytochrome C , and, except for the oxidation state of the iron, in myo globin and hemoglobin. 413
414
BIOINORGANIC CHEMISTRY
The peroxidatic activity of H R P is the catalysis of reactions by hydrogen peroxide and certain other oxidizing agents ( I ) summarized i n the following reaction scheme for H 0 and the substrate ferrocyanide. 2
HRP + H 0 2
2
&lapp
-+ Compound I
2
&2app
Compound I + ferrocyanide —* Compound II + ferricyanide &3app
Compound II + ferrocyanide —* H R P + ferricyanide
CH=CH
2
H C^Y
C H
3
HC^
-OOCCH CH 2
Figure 1.
3
-^^-CH=CH
\etni)
2
CH
2
^CH
CH CH COO' 2
2
Ferriprotoporphyrin IX or hemin
The ferrocyanide may be replaced by hydrogen donors such as p-cresol, i n which case free radicals are produced. Compounds I and II, which have unique spectral and chemical properties (2, 3, 4), were first observed by Theorell (5) and Keilin and M a n n (6). Compound I was found by Chance to contain two oxidizing equivalents in excess of that present in H R P (7) and Compound II by George to have one oxidizing equivalent (8). The stoichiometrics and reaction sequence have been verified (9) and early work has been well reviewed (1,9, 10). The formation of Compound I occurs with a rate constant, fci , of 1 X 1 0 M sec" and is pH-independent over a considerable range (11). In addition, equilibrium studies of ligand binding by native H R P indicated a p H independence of the binding constants i n some cases if the acid form of the ligand were assumed to be the binding species (10). The arguments concerning the interpretation of equilibrium ligand binding data app
1
7
_ 1
19.
Horseradish Peroxidase
DUNFORD AND HASINOFF
415
have been well summarized by B r i l l (10). These results, perhaps coupled with knowledge that the transition metal ion plays a role in the mechanism which could therefore be different from that for pure protein enzymes, have led to a widespread acceptance that H R P reactions are pH-independent, a reasonable conclusion on the basis of available evidence.
Fluoride and Cyanide Binding Kinetics by Native
HRP
W e undertook a kinetic study of fluoride binding by H R P i n collaboration with Robert Alberty while one of us ( H . B . D . ) was spending a sabbatical leave at the University of Wisconsin. The problem appeared to be simple and was ideally suited for the temperature-jump technique. Details of the kinetic results are published elsewhere (12, 13) but the simplest mechanism which is in accord with the kinetic data and does not violate Onsagers principle of detailed balancing (14) is
H P + F - «=> H P F 2
Ki
2
ft
ÎI
Ku
H P + F -
