Pantothenate Kinase Regulation of the Intracellular Concentration of Coenzyme A

Charles O Rock,Robert B Calder,Mohammad A Karim,Suzanne Jackowski

doi:10.1074/jbc.275.2.1377

Abstract

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway in bacteria and is thought to play a similar role in mammalian cells. We examined this hypothesis by identifying and characterizing two murine cDNAs that encoded PanK. The two cDNAs were predicted to arise from alternate splicing of the same gene to yield different mRNAs that encode two isoforms (mPanK1alpha and mPanK1beta) with distinct amino termini. The predicted protein sequence of mPanK1 was not related to bacterial PanK but exhibited significant similarity to Aspergillus nidulans PanK. mPanK1alpha was most highly expressed in heart and kidney, whereas mPanK1beta mRNA was detected primarily in liver and kidney. Pantothenate was the most abundant pathway component (42.8%) in normal cells providing clear evidence that pantothenate phosphorylation was a rate-controlling step in CoA biosynthesis. Enhanced mPanK1beta expression eliminated the intracellular pantothenate pool and triggered a 13-fold increase in intracellular CoA content. mPanK1beta activity in vitro was stimulated by CoA and strongly inhibited by acetyl-CoA illustrating that differential modulation of mPanK1beta activity by pathway end products also contributed to the management of CoA levels. These data support the concept that the expression and/or activity of PanK is a determining factor in the physiological regulation of the intracellular CoA concentration.

Highlights

Pantothenate kinase (PanK)1 (ATP:D-pantothenate 4Ј-phosphotransferase, EC 2.7.1.33) catalyzes the first committed step in the universal biosynthetic pathway leading to CoA
The first identification of cDNAs encoding mammalian PanK has enabled us to test the hypothesis that PanK expression can govern the intracellular CoA level
Whereas the supply of extracellular pantothenate (2 ␮M) was sufficient to maintain a detectable pool of intracellular pantothenate in the control cultures, pantothenate may have been limiting to CoA production in the pmPanK1␤-transfected cultures (Figs. 5 and 6)

Summary

Introduction

Pantothenate kinase (PanK) (ATP:D-pantothenate 4Ј-phosphotransferase, EC 2.7.1.33) catalyzes the first committed step in the universal biosynthetic pathway leading to CoA (for review see Ref. 1). PanK is the rate-controlling enzyme in CoA biosynthesis in Escherichia coli [1]. Nonesterified CoA is the most potent inhibitor of bPanK in vitro and in vivo, whereas acetylCoA is about 20% as effective as CoA [16] This feedback regulation is a primary mechanism by which bacteria control the cellular CoA level. PanK is proposed to be the master regulator of CoA biosynthesis in mammalian cells (for review see Ref. 17). Acetyl-CoA is significantly more effective than CoA at inhibiting mammalian PanK in cell extracts and partially purified preparations. CoA content, and to determine if CoA or acetyl-CoA is the most important feedback regulator of mammalian PanK activity

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Conclusion