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initial one. the Michaclis-Mcntcn cquation [13] in its normaI form is not valid. and a different hypcrbola dcscribes v as a lunetion of the total substratc concentration [14].

The aim of this paper is to show that this hypcrbola can be lincarizcd by a new method to obtain the enzyme fundamental constants (l'_mM and K_m). the total enzyme concentration and the catalytic constant. This lincarization method was employed to dctcrminc the kinctic parameters of chloropcroxidasc under conditions of Iow catalytic activity.

2. Kxperimental procedures

2.1. Materials

Chloropcroxidasc from Caldariomyces fumago [EC 1.11.1.10] (II000 U/ml) and fe/v-butylhydropcroxidc (70% vN in water) wcrc supplied by Fluka and wcrc uscd without further purification. Monochlorodimcdonc was a Sigma Chemical product and was uscd without (iirthcr purification. AU other reagents uscd wcrc of analytical gradc.

2.2. Kinetic measurements

The assay mixturc of chIoroperoxidasc consistcd of: potassium chloridc (0.55 mmol). /m-butylhydropcroxidc (0.9 pmol), a fixed amount of chloropcroxidasc (176 U), and various conccntrations of monochlorodimcdonc (110 ^?|7.7 -10 ^h) in a total volumc of 27.4 ml of potassium phosphatc buffer (0.1 M. pil 2.75).

The ratę of chloropcroxidasc-catalyzcd rcaction was measured in a controllcd quartz celi of 10 cm path-length at 25 ^CC by monitoring the dccrcasc of absorbancc at 278 nm duc to the convcrsion of monochlorodimcdonc (MCD. e₂7g"l-22 • 10⁴ M ¹ cm ') to dichlorodimcdonc (DCD, c₂₇₈= 1.6• 10² M”¹ cm ') [15]. 200 pi of the commcrcial enzyme was added to 2.5 ml ofO.l M phosphatc buffer. pH 6. aft er which 200 pl of this solution was taken and added to 0.1 M phosphate buffer pH 6 containing 20 mM KCI and 33 pM rm-butylhydropcroxidc. tBuOOH (finał volumc 27.4 ml). At pH 6 the enzyme activity was found to be stablc. Ali kinctic measurements wcrc perfoimcd in triplicatc.

Further. the UV spectrum of chloropcroxidasc was rccordcd in the abscncc of the substrates. monochlorodime-donc. and the oxidant. rm-butylhydropcroxidc.

3. Theory 3.1. Steady-state et/uation ofLaidler

A singlc-substratc system, assuming the abscncc of effeets of activators and inhibitora, is represented by the Michaclis-Mcntcn schcme [16.17] (sec Schcmc I). whcrc E, S. ES and P represent enzyme. substratc. cnzymc-substratc complex and product. respcctively. [E]₀ and [S]₀ indicatc initial conccntrations of enzyme and of substratc. From the Michaclis-Mcntcn schcme of rcaction. in 1955. Laidlcr [19] proposcd the following generał steady-state cquation:

[ES] =

*₂+*_, +*, -([E]₀ + [S]₀ - [P])± [*:+*-. +*. • ((E]₀ + (S]₀ - [P])] -JI -

2k_t

4-*Me]₀-((S]₀ [P])

[^•FA--,-fAv([E]o-F[S]₀-[E])]^;

0)

where [ES] depends on the total substratc concentration. The ratę cquation which can be casily obtaincd from laidlcr*s formulation (i^k.z [ES]) contains (besides the F,„,_x and K_m valucs) the total enzyme concentration [E]<> and the microscopic ratę constant k₂ of the Michaclis-Mcntcn rcaction schcmc. In the Michaclis-Mcntcn cquation. the ratc constant is inscparably linkcd with the cxprcssion (• [EJo^c*,* [E]o)-

3.2. Linearization method of generał steady-state et/nalion

Bccausc under the conditions dcscribcd, the known lincar plot arc not applicablc [20-23], for the estimation of Y_nixx. k₂ and [E]₀ valucs in the generał ratę equation. we proposc herc a lincarization method, which can be uscd to dctcrminc the enzyme constants.

P

[P]

E + S ^-

*t

[E]₀-[ES] [S]₀-[ES]-[P]

ES -—► E +

[ES]    [Elo-IES]

Schcnw I. Michaelis-Menten scheme for one-subslrale enz> me rcaction; steady stale conoenlrations of E, S, ES and P arc indicated.