HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hung, H.-C. Articles by Chang, G.-G. Search for Related Content PubMed PubMed Citation Articles by Hung, H.-C. Articles by Chang, G.-G. Social Bookmarking                   What's this?

Differentiation of the slow-binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase

Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China

Reprint requests to: Dr. Hui-Chih Hung, Department of Biochemistry, National Defense Medical Center, P.O. Box 90048-501 (Neihu), Taipei 114, Taiwan, Republic of China; e-mail: ibio33{at}ndmctsgh.edu.tw; fax: 886-2-2933-9996.

The binding mechanism of Mg²⁺ at the M3 site of human placental alkaline phosphatase was found to be a slow-binding process with a low binding affinity (K_Mg(app.) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg²⁺-free and Mg²⁺-containing enzymes by acrylamide showed almost identical dynamic quenching constant (K_sv = 4.44 ± 0.09 M^-1), indicating that there is no gross conformational difference between the M3-free and the M3-Mg²⁺ enzymes. However, Zn²⁺ was found to have a high affinity with the M3 site (K_Zn(app.) = 0.11 mM) and was observed as a time-dependent inhibitor of the enzyme. The dependence of the observed transition rate from higher activity to lower activity (k_obs) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational change of the enzyme, which locks the enzyme in a conformation (M3'-Zn) having an extremely high affinity for the Zn²⁺ (K*_Zn(app.) = 0.33 µM). The conformation of the M3'-Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg²⁺ activation and Zn²⁺ inhibition of the enzyme are reversible processes. Structural information indicates that the M3 site, which is octahedrally coordinated to Mg²⁺, has been converted to a distorted tetrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching constant for acrylamide (K_sv = 3.86 ± 0.04 M^-1), suggesting a conformational change. Both Mg²⁺ and phosphate prevent the enzyme from reaching this inactive structure. GTP plays an important role in reactivating the Zn-inhibited enzyme activity. We propose that, under physiological conditions, magnesium ion may play an important modulatory role in the cell for protecting the enzyme by retaining a favorable geometry of the active site needed for catalysis.

Keywords: Slow binding; magnesium ion; zinc ion; enzyme regulation; alkaline phosphatase

Abbreviations: GdmCl, guanidinium chloride • M3-Mg enzyme (M1,M2-Zn/M3-Mg), native human placental alkaline phosphatase with the M1 and M2 metal sites occupied by Zn²⁺ and M3 site by Mg²⁺ • M3-free enzyme (M1,M2-Zn/M3-free), the enzyme with the M1 and M2 metal sites occupied by Zn²⁺ but M3 site is free • M3'-Zn enzyme (M1,M2,M3'-Zn), inactive enzyme with all metal sites occupied by Zn²⁺ • M1',M2',M3-free enzyme, inactive metal-free apo-enzyme with all metal sites free • M1',M2',M3-Mg, reconstituted enzyme with M3 site occupied by Mg²⁺ but the M1 and M2 sites are free • M1',M2'-Zn/M3-Mg, reconstituted fully charged metal ions but partially active enzyme because of altered M1' and M2' zinc sites.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				I. Benzaghou, I. Bougie, and M. Bisaillon Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase J. Biol. Chem., November 26, 2004; 279(48): 49755 - 49761. [Abstract] [Full Text] [PDF]

				H.-C. Chang, W.-Y. Chou, and G.-G. Chang Effect of Metal Binding on the Structural Stability of Pigeon Liver Malic Enzyme J. Biol. Chem., February 8, 2002; 277(7): 4663 - 4671. [Abstract] [Full Text] [PDF]