title: Evidence for hysteretic substrate channeling in the proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase coupled reaction of proline utilization A (PutA).
abstract: PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ(1)-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 Δ(1)-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-γ-semialdehyde (the hydrolyzed form of Δ(1)-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD(+)-producing NADH and 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.
authors.affiliation: From the Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 and.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Moxley
authors.forename: Nikhilesh
authors.initials: N
authors.lastname: Sanyal
authors.forename: Navasona
authors.initials: N
authors.lastname: Krishnan
authors.forename: John J
authors.initials: JJ
authors.lastname: Tanner
authors.forename: Donald F
authors.initials: DF
authors.lastname: Becker
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009243:NAD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011394:Proline Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D009243:NAD; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011394:Proline Oxidase
keywords: Enzyme Kinetics; Enzyme Mechanisms; Enzyme Turnover; Flavin; Hysteresis; Metabolism; Proline; Substrate Channeling