Further Analysis of Maize C 4 Pyruvate,Orthophosphate Dikinase Phosphorylation by Its Bifunctional Regulatory Protein Using Selective Substitutions of the Regulatory Thr-456 and Catalytic His-458 Residues 1 Chris J. Chastain,* ,2 Montgomery Botschner,* Grant E. Harrington,* Brent J. Thompson,* ,3 Sarah E. Mills,* Gautam Sarath,† and Raymond Chollet† *Department of Biology, Moorhead State University, Moorhead, Minnesota 56563; and †George W. Beadle Center, Department of Biochemistry, University of Nebraska—Lincoln, Lincoln, Nebraska 68588-0664 Received September 15, 1999, and in revised form November 15, 1999 In C 4 plants such as maize, pyruvate,orthophosphate dikinase (PPDK) catalyzes the regeneration of the ini- tial carboxylation substrate during C 4 photosynthesis. The primary catalytic residue, His-458 (maize C 4 PPDK), is involved in the ultimate transfer of the -phosphate from ATP to pyruvate. C 4 PPDK activity undergoes light– dark regulation in vivo by reversible phosphorylation of a nearby active-site residue (Thr- 456) by a single bifunctional regulatory protein (RP). Using site-directed mutagenesis of maize recombinant C 4 dikinase, we made substitutions at the catalytic His residue (H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of these affinity-purified mutant enzymes was assayed for changes in dikinase activity. As expected, substituting His-458 with Asn results in a catalytically incompetent enzyme. Substi- tutions of the Thr-456 residue with Tyr and Phe re- duced activity by about 94 and 99%, respectively. In- sertion of Glu at this position completely abolished activity, presumably by the introduction of negative charge proximal to the catalytic His. Furthermore, neither the T456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP. The inability of the former to serve as a phosphorylation substrate indi- cates that RP is functionally a member of the Ser/Thr family of protein kinases rather than a “dual-specific- ity” Ser-Thr/Tyr kinase, since our previous work showed that RP effectively phosphorylated Ser in- serted at position 456. The inability of RP to phosphor- ylate its native target Thr residue when Asn is substi- tuted for His-458 documents that RP requires the His-P catalytic intermediate form of PPDK as its pro- tein substrate. For these latter studies, synthetic phos- phopeptide-directed antibodies specific for the Thr 456 -P form of maize C 4 PPDK were developed and characterized. © 2000 Academic Press Key Words: pyruvate, Pi dikinase (PPDK); site-di- rected mutagenesis; protein phosphorylation; regula- tory protein; C 4 photosynthesis; maize (Zea mays). A key regulatory enzyme of the C 4 -photosynthetic pathway is pyruvate,orthophosphate dikinase (PPDK 4 ; EC 2.7.9.1) (1–3). In C 4 leaves, PPDK is localized pre- dominantly in the chloroplast stroma of mesophyll cells, where it is active as a homotetramer of 95-kDa subunits. The enzyme catalyzes the regeneration of the primary CO 2 acceptor, phosphoenolpyruvate (PEP), for PEP carboxylase from pyruvate and ATP/Pi in a three- step catalytic reaction sequence (4, 5): Pyruvate + ATP + Pi 7 PEP + AMP + PPi. 1 This work was supported in part by Grant NRICGP/USDA 9703445 (to C.J.C.), a Council on Undergraduate Research/Merck Company Foundation Summer Research Fellowship (to B.J.T.), NSF Grant MCB-9727236 (to R.C.), and the Center for Biotechnology, University of Nebraska—Lincoln, funded through the Nebraska Re- search Initiative (G.S.). 2 To whom correspondence should be addressed. Fax: 1-218-236- 2018. E-mail: chastain@mhd1.moorhead.msus.edu. 3 Present address: The Center for Molecular Neuroscience, Vanderbilt University, Nashville, TN 37232. 4 Abbreviations used: BSA, bovine serum albumin; DTT, dithio- threitol; FPLC, fast protein liquid chromatography; PEP, phos- phoenolpyruvate; PMSF, phenylmethylsulfonyl fluoride; PPDK, pyruvate,orthophosphate dikinase; RP, regulatory protein; TBS, Tris-buffered saline. 0003-9861/00 $35.00 165 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 375, No. 1, March 1, pp. 165–170, 2000 doi:10.1006/abbi.1999.1651, available online at http://www.idealibrary.com on