1AJA

THREE-DIMENSIONAL STRUCTURE OF THE D153G MUTANT OF E. COLI ALKALINE PHOSPHATASE: A MUTANT WITH WEAKER MAGNESIUM BINDING AND INCREASED CATALYTIC ACTIVITY

Summary for 1AJA

• Entry DOI: 10.2210/pdb1aja/pdb
• Descriptor: ALKALINE PHOSPHATASE (1 entity in total)
• Functional Keywords: non specific mono-esterase
• Biological source: Escherichia coli
• Cellular location: Periplasm: P00634
• Total number of polymer chains: 2
• Total formula weight: 94074.70

Authors

Dealwis, C.G.,Chen, L.,Abad-Zapatero, C. (deposition date: 1995-08-19, release date: 1995-11-14, Last modification date: 2024-10-09)

Primary citation

Dealwis, C.G.,Brennan, C.,Christianson, K.,Mandecki, W.,Abad-Zapatero, C.
Crystallographic analysis of reversible metal binding observed in a mutant (Asp153-->Gly) of Escherichia coli alkaline phosphatase.
Biochemistry, 34:13967-13973, 1995

PubMed Abstract: Here we present the refined crystal structures of three different conformational states of the Asp153-->Gly mutant (D153G) of alkaline phosphatase (AP), a metalloenzyme from Escherichia coli. The apo state is induced in the crystal over a 3 month period by metal depletion of the holoenzyme crystals. Subsequently, the metals are reintroduced in the crystalline state in a time-dependent reversible manner without physically damaging the crystals. Two structural intermediates of the holo form based on data from a 2 week (intermediate I) and a 2 month soak (intermediate II) of the apo crystals with Mg2+ and Zn2+ have been identified. The three-dimensional crystal structures of the apo (R = 18.1%), intermediate I (R = 19.5%), and intermediate II (R = 19.9%) of the D153G enzyme have been refined and the corresponding structures analyzed and compared. Large conformational changes that extend from the mutant active site to surface loops, located 20 A away, are observed in the apo structure with respect to the holo structure. The structure of intermediate I shows the recovery of the entire enzyme to an almost native-like conformation, with the exception of residues Asp 51 and Asp 369 in the active site and the surface loop (406-410) which remains partially disordered. In the three-dimensional structure of intermediate II, both Asp 51 and Asp 369 are essentially in a native-like conformation, but the main chain of residues 406-408 within the loop is still not fully ordered. The D153G mutant protein exhibits weak, reversible, time dependent metal binding in solution and in the crystalline state.(ABSTRACT TRUNCATED AT 250 WORDS)

PubMed: 7577993
DOI: 10.1021/bi00043a001

Experimental method

X-RAY DIFFRACTION (2.5 Å)