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Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.

Ma L, Tibbitts TT, Kantrowitz ER
Protein Sci 4 1498-506 (1995)
Cited: 15 times
EuropePMC logo PMID: 8520475

Abstract

The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)

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  2. High-resolution analysis of Zn(2+) coordination in the alkaline phosphatase superfamily by EXAFS and x-ray crystallography. Bobyr E, Lassila JK, Wiersma-Koch HI, Fenn TD, Lee JJ, Nikolic-Hughes I, Hodgson KO, Rees DC, Hedman B, Herschlag D. J Mol Biol 415 102-117 (2012)
  3. Role of metal ions on the secondary and quaternary structure of alkaline phosphatase from bovine intestinal mucosa. Bortolato M, Besson F, Roux B. Proteins 37 310-318 (1999)
  4. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site. Sunden F, Peck A, Salzman J, Ressl S, Herschlag D. Elife 4 (2015)
  5. Prediction of distal residue participation in enzyme catalysis. Brodkin HR, DeLateur NA, Somarowthu S, Mills CL, Novak WR, Beuning PJ, Ringe D, Ondrechen MJ. Protein Sci 24 762-778 (2015)
  6. Characterization of heterodimeric alkaline phosphatases from Escherichia coli: an investigation of intragenic complementation. Hehir MJ, Murphy JE, Kantrowitz ER. J Mol Biol 304 645-656 (2000)
  7. Ternary zinc(II)-dipeptide complexes for the hydrolytic cleavage of DNA at physiological pH. Reddy PR, Mohan SK, Rao KS. Chem Biodivers 2 672-683 (2005)
  8. Cavity scaling: automated refinement of cavity-aware motifs in protein function prediction. Chen BY, Bryant DH, Fofanov VY, Kristensen DM, Cruess AE, Kimmel M, Lichtarge O, Kavraki LE. J Bioinform Comput Biol 5 353-382 (2007)
  9. Kinetics of complexing activation by the magnesium ion on green crab (Scylla serrata) alkaline phosphatase. Park YD, Yang Y, Chen QX, Lin HN, Liu Q, Zhou HM. Biochem Cell Biol 79 765-772 (2001)
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  14. Slipknotted and unknotted monovalent cation-proton antiporters evolved from a common ancestor. Zayats V, Perlinska AP, Jarmolinska AI, Jastrzebski B, Dunin-Horkawicz S, Sulkowska JI. PLoS Comput Biol 17 e1009502 (2021)


Related citations provided by authors (2)

  1. Reaction Mechanism of Alkaline Phosphatase Based on Crystal Structures. Two Metal Ion Catalysis. Kim EE, Wyckoff HW J. Mol. Biol. 218 449- (1991)
  2. Mutations at histidine 412 alter zinc binding and eliminate transferase activity in Escherichia coli alkaline phosphatase.. Ma L, Kantrowitz ER J Biol Chem 269 31614-9 (1994)

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