Role of conformational change in the fumarase reaction cycle.
Activation of fumarase by high concentrations of either malate or fumarate, often referred to as negative cooperativity, can be explained without assuming additional sites of substrate action or subunit-subunit interactions. The following observations support a model based on a rate-dependent recycling of free enzyme through a sequence of conformational states that differ in substrate specificity and catalytic activity: (1) Displacement from equilibrium of a radiolabeled malate/fumarate probe is readily induced by moderate concentrations of either substrate. This phenomenon, called substrate-induced countertransport, indicates that the steady-state ratio of free enzyme forms is very dependent on substrate concentration. (2) Related to this, the back-labeling that can be observed with either 14C product with either substrate in the steady state is more rapid than expected for a single free enzyme state model. (3) Fumarate, more strongly than malate, shows competitive effects as a product. This may reflect a higher affinity of fumarate for an isoform that also reacts with malate. (4) P(i), an activator of fumarase at midrange substrate concentration, overcomes strong competitive inhibition by fumarate of the M-->F reaction and increases recycling as shown by its effect on counterflow. To the extent that these effects are due to buffer activation, they suggest that proton transfer between solvent and the enzyme site is important in determining the recycling rate. (5) Transaconitate, a competitive inhibitor, overcomes counterflow induced by either substrate, indicating that recycling events occur in the enzyme-transaconitate complex.
Rose
I A
IA
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Warms
J V
JV
Yuan
R G
RG
D004789:Enzyme Activation; D005649:Fumarate Hydratase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008293:Malates / Q000494:pharmacology; D008433:Mathematics; D008962:Models, Theoretical; D011487:Protein Conformation
D008293:Malates; D005649:Fumarate Hydratase