Zinc-, cobalt- and iron-chelated forms of adenylate kinase from the Gram-negative bacterium Desulfovibrio gigas.
Adenylate kinase (AK) from the sulphate-reducing bacterium Desulfovibrio gigas (AK) has been characterized earlier as a Co(2+)/Zn(2+)-containing enzyme, which is an unusual characteristic for adenylate kinases from Gram-negative bacteria, in which these enzymes are normally devoid of metal ions. AK was overexpressed in E. coli and homogeneous Co(2+)-, Zn(2+)- and Fe(2+)-forms of the enzyme were obtained under in vivo conditions. Their structural stability and spectroscopic and kinetic properties were compared. The thermal denaturation of Co(2+)- and Zn(2+)-forms of AK was studied as a cooperative two-state process, sufficiently reversible at pH 10, which can be correctly interpreted in terms of a simple two-state thermodynamic model. In contrast, the thermally induced denaturation of Fe(2+)-AK is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Practically identical contents of secondary-structure elements were found for all the metal-chelated-forms of AK upon analysis of circular dichroism data, while their tertiary structures were significantly different. The peculiar tertiary structure of Fe(2+)-AK, in contrast to Co(2+)- and Zn(2+)-AK, and the consequent changes in the physico-chemical and enzymatic properties of the enzyme are discussed.
REQUIMTE, Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
Anna V
AV
Kladova
Olga Yu
OY
Gavel
Galina G
GG
Zhadan
Manuel G
MG
Roig
Valery L
VL
Shnyrov
Sergey A
SA
Bursakov
D002614:Chelating Agents / Q000494:pharmacology*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003035:Cobalt / Q000737:chemistry*; D045286:Desulfovibrio gigas / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006090:Gram-Negative Bacteria / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013053:Spectrophotometry / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry*
D002614:Chelating Agents; D003035:Cobalt; D007501:Iron; D015032:Zinc