Electrostatic channeling of substrates between enzyme active sites: comparison of simulation and experiment.
Recent simulation work has indicated that channeling of charged substrates between the active sites of bifunctional enzymes or bienzyme complexes can be significantly enhanced by favorable interactions with the electrostatic field of the enzymes. The results of such simulations are expressed in terms of transfer efficiencies, which describe the probability that substrate leaving the active site of the first enzyme will reach the active site of the second enzyme before escaping out into bulk solution. The experimental indicators of channeling, on the other hand, are factors such as a decrease in the transient (lag) time for appearance of the final product of the coupled enzyme reaction or a decrease in the susceptibility of the overall reaction rate to the presence of competing enzymes or competitive inhibitors. The work reported here aims to establish a connection between the transfer efficiencies obtained from simulation, with the above-mentioned experimental observables. This is accomplished by extending previously reported analytical approaches to combine the simulated transfer efficiency with the Michaelis-Menten kinetic parameters Km and Vmax of the enzymes involved; expressions are derived to allow both transient times and steady state rates to be calculated. These results are applied to the two systems that have been studied both theoretically and experimentally. In the first case, that of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS), the experimentally observed decrease in transient times is found to be consistent with a transfer efficiency of >/=80%. In the second case, that of a citrate synthase-malate dehydrogenase fusion protein, a transfer efficiency of 73% is consistent with the experimental transient time measurements. Separate and independent analysis of the effects of adding the competing enzyme aspartate aminotransferase gives a transfer efficiency of 69%, in excellent agreement with the transient time results. The transfer efficiencies thus obtained from experimental results are in both cases in good agreement with those obtained from simulations that include electrostatic interactions. One important discrepancy between simulation and experiment, is however, found in the reported effects of adding a competitive inhibitor in the DHFR-TS system: qualitatively different results are expected from the theoretical analysis. A possible reason for this apparent contradiction is discussed.
Elcock
A H
AH
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0365, USA.
Huber
G A
GA
McCammon
J A
JA
D001219:Aspartate Aminotransferases / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000378:metabolism; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055672:Static Electricity; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000378:metabolism
D004791:Enzyme Inhibitors; D004798:Enzymes; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C056930:thymidylate synthase-dihydrofolate reductase; D013938:Thymidine Monophosphate; D008291:Malate Dehydrogenase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D002950:Citrate (si)-Synthase; D001219:Aspartate Aminotransferases