Evidence for Hysteretic substrate channeling in the Proline Dehydrogenaseandδ¹-Pyrroline-5-carboxylate Dehydrogenase coupled reaction of Proline UtilizationA(PutA)

Background: PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism. Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step. Conclusion: Substrate channeling in PutA exhibits hysteretic behavior. Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation. PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and δ¹-pyrroline- 5-carboxylate dehydrogenase (P5CDH) domains that catalyze the oxidation of L-proline to L-glutamate in two successive reactions. In the PRODH active site, proline undergoes a two-electron oxidation to δ¹-pyrroline-5- carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, L-glutamate- semialdehyde (the hydrolyzed form ofδ¹-pyrroline- 5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred toNAD+-producingNADHand glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput.We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed.