Pyrococcus abyssi alkaline phosphatase: the dimer is the active form.
Alkaline phosphatases (APs), E.C. 3.1.3.1, are non-specific phosphomonoesterases optimally active under alkaline conditions. They are classically known to be homodimeric metalloenzymes. This quaternary structure has been considered necessary for activity, although the relationship between quaternary structure and activity is not well understood. Recombinant Pyrococcus abyssi AP was previously isolated and characterized, appearing to have two active quaternary structures on native polyacrylamide gel electrophoresis: a monomer and a homodimer. The purpose of the present work was to determine the actual quaternary structure of P. abyssi AP in solution, by isolating each of the two quaternary forms and establishing the parameters governing the assembly and dissociation of the dimer. pH appeared to be an important parameter: in acidic media, the monomer/dimer ratio shifted towards monomer. Buffer composition also affected the quaternary structure: at the same pH, in potassium phosphate buffer, the two quaternary structures were observed, whereas in tris(hydroxymethyl)aminomethane hydrochloride buffer, only the dimer was observed. Metals bound to the enzyme were found to be involved in the stability of the quaternary structure. Indeed, the P. abyssi AP obtained upon removal of the metals was monomeric. Reactivation of the latter was achieved with variable efficiency. From these experiments, no active monomer could be isolated, leading the conclusion that the active form of P. abyssi AP is the homodimer.
Zappa
S
S
Laboratoire des Sciences du Génie Chimique, CNRS, INPL-ENSAIA, 2 avenue de la Forêt de Haye, B.P. 172, 54505 Vandoeuvre-lès-Nancy Cedex, France. szappa@cea.fr
Boudrant
J
J
Kantrowitz
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ER
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D019281:Dimerization; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D019711:Pyrococcus / Q000201:enzymology*; D013329:Structure-Activity Relationship
D008670:Metals; D000469:Alkaline Phosphatase