Tài liệu Báo cáo Y học: Ligand interactions and protein conformational changes of

Ligand interactions and protein conformational changes

of phosphopyridoxyl-labeled Escherichia coli phosphoenolpyruvate

carboxykinase determined by fluorescence spectroscopy

Marı´a Victoria Encinas1

, Fernando D. Gonza´ lez-Nilo1

, Hughes Goldie2 and Emilio Cardemil1

1

Departamento de Ciencias Quı´micas, Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile, Chile; 2

Department of

Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada

Escherichia coli phosphoenolpyruvate (PEP) carboxykinase

catalyzes the decarboxylation of oxaloacetate and transfer of

the c-phosphoryl group of ATP to yield PEP,ADP,and

CO2. The interaction of the enzyme with the substrates ori￾ginates important domain movements in the protein. In this

work,the interaction of several substrates and ligands with

E. coli PEP carboxykinase has been studied in the phos￾phopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatized

enzyme retained the substrate-binding characteristics of the

native protein,allowing the determination of several pro￾tein–ligand dissociation constants,as well as the role of

Mg2+ and Mn2+ in substrate binding. The binding affinity

of PEP to the enzyme–Mn2+ complex was )8.9 kcalÆmol)1

,

which is 3.2 kcalÆmol)1 more favorable than in the complex

with Mg2+. For the substrate nucleotide–metal complexes,

similar binding affinities ()6.0 to )6.2 kcalÆmol)1

) were

found for either metal ion. The fluorescence decay of the

P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%)

and 1.2 ns. These lifetimes were markedly altered in the

derivatized enzyme–PEP–Mn complexes,and smaller

changes were obtained in the presence of other substrates.

Molecular models of the P-pyridoxyl–E. coli PEP carb￾oxykinase showed different degrees of solvent-exposed sur￾faces for the P-pyridoxyl group in the open (substrate-free)

and closed (substrate-bound) forms,which are consistent

with acrylamide quenching experiments,and suggest that the

fluorescence changes reflect the domain movements of the

protein in solution.

Keywords: Escherichia coli phosphoenolpyruvate carboxy￾kinase; ligand binding; conformational changes; P-pyridoxyl

fluorescence spectroscopy.

Escherichia coli phosphoenolpyruvate carboxykinase [PEP

carboxykinase; ATP:oxaloacetate carboxylase (trans-phos￾phorylating) EC 4.1.1.49] catalyzes the reversible decarb￾oxylation of oxaloacetic acid (OAA) with the associated

transfer of the c-phosphoryl group of ATP to yield PEP and

ADP,where M2+ is a divalent metal ion:

OAA þ ATP !

M2þ

PEP þ ADP þ CO2

The physiological role of this enzyme in bacteria and most

other organisms is to catalyze the formation of PEP in the

first committed step of gluconeogenesis [1]. The crystal

structure of free- and substrate-bound E. coli PEP carb￾oxykinase has been solved at 1.9 A˚ resolution [2,3]. The

enzyme is a monomeric,globular protein that belongs to the

a/b protein class. The overall structure has two domains,a

275 residue N-terminal domain,and a more compact 265

residue C-terminal domain,with the active site in a deep

cleft between them. The recently reported crystal structure

of Trypanosoma cruzi PEP-carboxykinase [4] shows

remarkable similarity. Upon substrate binding,the E. coli

enzyme undergoes a domain closure through a 20
rotation

of the two domains towards each other,excluding bulk

solvent from the active site and positioning active site

residues for catalysis [3]. Results obtained with AlF3

complexes of E. coli PEP carboxykinase indicate that

phosphoryl transfer occurs via a direct displacement mech￾anism with associative qualities [5]. In spite of the detailed

knowledge of the structural characteristics of E. coli carb￾oxykinase,very little information is available for ATP￾dependent carboxykinases with respect to thermodynamic

data on ligand binding [6].

Chemical modification studies have shown that PLP

specifically labels the protein in a lysyl residue located at

position 288 and,upon reduction of the labeled enzyme with

sodium borohydride,a P-pyridoxyl group is covalently

attached at this site [7]. The crystal coordinates of the E. coli

enzyme indicate that this residue,located in the C-terminal

domain,is 9.7 A˚ from Gly251,which is the closest amino

acid residue of the N-terminal domain,in the P-loop of the

enzyme. Upon domain closure,the distance from Lys288 to

Gly251 reduces to 5.3 A˚ ,thus making Lys288 an excellent

observation point to follow the domain movement of the

protein in solution,provided this motion can be detected.

Spectroscopic properties of the Schiff base formed upon

reaction of PLP with amino acids or amines are highly

dependent on medium properties such as pH or polarity

[8,9]. Spectroscopic studies have been employed to obtain

information about the mechanism of some PLP-dependent

enzymes [10]. Reduction of the imine bond with NaBH4

Correspondence to M. V. Encinas,Departamento de Ciencias

Quı´micas,Facultad de Quı´mica y Biologı´a,Universidad

de Santiago de Chile,Casilla 40,Santiago 33,Chile.

Fax: + 56 2 681 2108,Tel.: + 56 2 681 2575;

E-mail: [email protected]

Abbreviations: OAA,oxaloacetic acid; PEP,phosphoenolpyruvate;

PLP,pyridoxal 5¢-phosphate; P-pyridoxyl,phosphopyridoxyl.

(Received 16 May 2002,revised 26 July 2002,

accepted 21 August 2002)

Eur. J. Biochem. 269,4960–4968 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03196.x