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Originally published In Press as doi:10.1074/jbc.M909488199 on March 22, 2000

J. Biol. Chem., Vol. 275, Issue 21, 15773-15781, May 26, 2000
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Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase*

Jason C. Baker, Xiaohua Yan, Tao Peng, Shane Kasten, and Thomas E. RocheDagger

From the Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506

Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 µM L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in kcat along with about a 2-fold decrease in the Km of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >= 3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.

* This work was supported by National Institutes of Health Grant DK18320 and by the Kansas Agriculture Experiment Station Contribution 00-189-J.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, 104 Willard Hall, Kansas State University, Manhattan, KS 66506. Tel.: 785-532-6116; Fax: 785-532-7278.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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