Biochimica et Biophysica Acta, 1119 (1992) 35-38 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00 35 BBAPRO 34089 ATP-dependent Saccharomyces cerevisiae phosphoenol pyruvate carboxykinase: isolation and sequence of a peptide containing a highly reactive cysteine Marysol Alvear 1, M. Victoria Encinas 2, Robert G. Kemp 3, Steven P. Latshaw 3 and Emilio Cardemil 2 ! Departamo~to de Ciencias Qalmicas, Facultad de bTgenierfa y Admbustracidn, Unicersidad de la Frontera, Temuco (Chile), 2 Departamento de Qufmica, Unirersidad de Santiago de Chile, Santiago (Chile) attd 3 Department of Biological Chemistry, The Chicago Medical School, North Chicago, IL ( U.S.A.) (Received 12 September 1991) Key words: Phosphoazolpyruvate carboxykinase; Nucleotide binding; Cysteine: (S. cerecisiae) Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (EC 4.1.1.49), inactivated by N-(iodoacetyl)-N'-(5- sulfo-l.naphthyl)ethylenediamine, incorporated 0.95 mol of the fluorescent moiety per moi of enzyme subunit. Reagent incorporation was completely protected by the presence of ADP plus MnCI 2. The labeled protein was digested with trypsin after carboxymethylation. Two labeled peptides were isolated by reverse-phase high-perfor- mance liquid chromatography and were sequenced by gas-phase automatic Edman degradation. Both peptides contained overlapping amino acid sequences from .Asn-358 to Lys-375, thus identifying Cys-364 as the reactive amino acid residue. The position of the target amino acid residue is immediately preceding a putative phosphoryl- binding sequence proposed for some nucleotide-binding proteins. Phosphoenolpyruvate carboxykinases (PEPCKs) (ATP/GTP: oxaloacetate carboxy-lyases (transphos- phorylating) EC 4.1.1.49/32) play an important role in the control of gluconeogenesis and, depending on the species, they require guanosine or adenosine nu- cleotides [1]. GTP-dependent enzymes are monomers of about 70 kDa found almost exclusively in animals, whereas ATP-dependent PEPCKs are usually (but not always) multisubunit proteins found in plants and mi- croorganisms (see Refs. 2-4 and the references cited therein). From studies carried out in recent years, it is apparent that ATP-dependent and GTP-dependent PEPCKs share significant amino acid sequence similar- ity within their respective classes but not between one Abbreviations: PEPCK, phosphoato/pyruvate carboxykinase; 1,5-I- AEDANS, N-(iodoacetyl)-N '-(5-sulfo- 1-naphthyl)et hylenediamine; Hepes, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; TPCK, L-l-tosylamido-2-phenylethyl chloromethyl ketone; HPLC, high-performance liquid chromatography; TFA, trifluoroacetic acid; PITC, phenylisothiocarbamoyl; PTH, phenylthiohydantoin. Correspondence: E. Cardemil, Departamento de Quimica, Universi- dad de Santiago de Chile, Casilla 307, Santiago-2, Chile. group and the other, except for short consensus se- quences for phosphoryl-binding regions [3,4]. The use of a number of group-selective reagents for the chemical modification of PEPCKs has indicated the presence of arginine [5,6], lysine [7] and histidine [8]. It is noteworthy that all carboxykinases studied thus far have been reported to be sensitive to inactivation by thiol-modifying reagents [2,9]. It has been reported that the rat liver enzyme has a pair of vicinal cysteines probably located in the GTP-binding site [10,11], but only one of them (Cys-288) is modified by the fluores- cent reagent N-(7-dimethylamino-4-methylcoumarin- yl)maleimide [9]. We have recently shown that Saccha- romyces cerevisiae PEPCK is rapidly inactivated by incubation with the sulfhydryl-specific reagent 1,5-1- AEDANS, and that complete inactivation is correlated with the incorporation of 4 mol of reagent per homote- tramer [12]. Notably, the presence of ADP plus MnCl 2 completely prevented both inactivation and reagent incorporation. In order to get further information about the func- tional residue modified by 1,5-I-AEDANS in yeast PEPCK [12], we have analyzed the specific residues labeled upon enzyme inactivation by this reagent. In