Kinetic Analysis of the Inhibition Mechanism of Bovine Mitochondrial F1-ATPase Inhibitory Protein Using Biochemical Assay

Overview

Journal J Biochem

Publisher Oxford University Press

Specialty Biochemistry

Date 2021 Mar 11

PMID 33693769

Citations 8

Authors

Ryohei Kobayashi

Sougo Mori

Hiroshi Ueno

Hiroyuki Noji

Affiliations

Soon will be listed here.

Abstract

ATPase inhibitory factor 1 (IF1) is a mitochondrial regulatory protein that blocks ATP hydrolysis of F1-ATPase, by inserting its N-terminus into the rotor-stator interface of F1-ATPase. Although previous studies have proposed a two-step model for IF1-mediated inhibition, the underlying molecular mechanism remains unclear. Here, we analysed the kinetics of IF1-mediated inhibition under a wide range of [ATP]s and [IF1]s, using bovine mitochondrial IF1 and F1-ATPase. Typical hyperbolic curves of inhibition rates with [IF1]s were observed at all [ATP]s tested, suggesting a two-step mechanism: the initial association of IF1 to F1-ATPase and the locking process, where IF1 blocks rotation by inserting its N-terminus. The initial association was dependent on ATP. Considering two principal rotation dwells, binding dwell and catalytic dwell, in F1-ATPase, this result means that IF1 associates with F1-ATPase in the catalytic-waiting state. In contrast, the isomerization process to the locking state was almost independent of ATP, suggesting that it is also independent of the F1-ATPase state. Further, we investigated the role of Glu30 or Tyr33 of IF1 in the two-step mechanism. Kinetic analysis showed that Glu30 is involved in the isomerization, whereas Tyr33 contributes to the initial association. Based on these findings, we propose an IF1-mediated inhibition scheme.

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