Lack of deviation from Michaelis–Menten kinetics for pig heart fumarase

Abstract

Studies of steady-state kinetics of fumarase in the usual substrate-concentration range from 0.1 Km to 10 Km and in the high substrate-concentration range from 10 Km to 200 Km are described. The purpose is to investigate reports of substrate inhibition and oscillatory kinetics. In the normal substrate-concentration range, no deviations from hyperbolic kinetics were found, and in the extended concentration range, up to more than 200 times the Km value, no substrate inhibition was demonstrated. A discussion of the discrepancies between the mentioned reports of deviations from the hyperbolic kinetics and the present findings is given.

Pig heart fumarase really does exhibit negative kinetic co-operativity at a constant ionic strength

“…1) an essentially straight-line plot is obtained. The last result is similar to that obtained by Andersen (1980) and Wharton & Szawelski (1982), and it leads to incorrect conclusions about the enzyme mechanism. The change in v due to the change in ionic strength is just sufficient to eliminate the curvature of the Lineweaver-Burk plot.…”

“…This conclusion seems to be at odds with the studies by Alberty and co-workers (Alberty & Bock, 1953;Alberty et a!., 1954;Taraszka & Alberty, 1964) and themore recent study by Bardsley et al (1980). Although Andersen (1980) and Wharton & Szawelski (1982) were careful to prepare substrate solutions of the correct pH, neither of these studies appears to have maintained a constant ionic strength as the substrate concentration was increased. From the results obtained by Frieden et al (1957) and Alberty & Hammes (1958) it is known that some of the fumarase kinetic parameters are quite sensitive to ionic strength, as might be expected for the reaction of a dianionic substrate.…”

“…Previous studies by Andersen (1980) and Wharton & Szawelski (1982) have indicated that the fumarasecatalysed interconversion of L-malate and fumarate does not deviate from simple Michaelis-Menten kinetics. This conclusion seems to be at odds with the studies by Alberty and co-workers (Alberty & Bock, 1953;Alberty et a!., 1954;Taraszka & Alberty, 1964) and themore recent study by Bardsley et al (1980).…”

“…From the results obtained by Frieden et al (1957) and Alberty & Hammes (1958) it is known that some of the fumarase kinetic parameters are quite sensitive to ionic strength, as might be expected for the reaction of a dianionic substrate. In order to resolve this discrepancy, we have performed experiments similar to those of Andersen (1980) where the ionic strength increased with increasing substrate concentration, and others where the ionic strength was maintained at a constant value by the addition of an inert electrolyte.…”

Pig Heart Fumarase Contains Two Distinct Substrate-binding Sites Differing in Affinity

“…Alternatively, the second substrate-binding sites may be potential catalytic sites as well. A number of anions, especially phosphate, have also been shown to affect the activity of pig heart fumarase (1,11,(17)(18)(19). They can be supposed to bind into the substrate-binding sites as well, thereby exerting an effect on the enzyme which is comparable to the effect induced by the substrates themselves.…”