1hqa
From Proteopedia
(New page: 200px<br /><applet load="1hqa" size="450" color="white" frame="true" align="right" spinBox="true" caption="1hqa, resolution 2.25Å" /> '''ALKALINE PHOSPHATASE...) |
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| - | [[Image:1hqa.jpg|left|200px]]<br /><applet load="1hqa" size=" | + | [[Image:1hqa.jpg|left|200px]]<br /><applet load="1hqa" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1hqa, resolution 2.25Å" /> | caption="1hqa, resolution 2.25Å" /> | ||
'''ALKALINE PHOSPHATASE (H412Q)'''<br /> | '''ALKALINE PHOSPHATASE (H412Q)'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Site-specific mutagenesis has been used to replace His-412 with glutamine | + | Site-specific mutagenesis has been used to replace His-412 with glutamine in Escherichia coli alkaline phosphatase. In the wild-type enzyme His-412 is a direct ligand to one of the catalytically important zinc atoms (Zn1) in the active site. The mutant enzyme (H412Q) exhibited about the same k(cat), but a 50-fold increase in K(m) compared to the corresponding kinetic parameters for the wild-type enzyme. Furthermore, the H412Q enzyme had a lower zinc content than the wild-type enzyme. In contrast to the wild-type enzyme, Tris was less effective in the transferase reaction and dramatically inhibited the hydrolysis reaction of the H412Q enzyme. The addition of zinc to the mutant enzyme increased the k(cat) value above that of the wild-type enzyme, partially restored the weak substrate and phosphate binding, and also alleviated the inhibition by Tris. The structure of the H412Q enzyme was also determined by X-ray crystallography. The overall structure of the H412Q enzyme was very similar to that of the wild-type enzyme; the only alpha-carbon displacements over 1 angstrom were observed near the mutation site. In the H412Q structure no phosphate was bound in the active site of the enzyme; however, two water molecules were observed where phosphate normally binds in the wild-type enzyme. Close examination of the active site of the H412Q structure revealed structural changes in Ser-102 as well as at the mutation site. For example, the carbonyl oxygen of the side chain of Gln-412 rotated away from the position of His-412 in the wild-type structure, although too far away (3.2 angstroms) to coordinate to Zn1. Studies on the H412Q enzyme, and a comparison of the H412Q and H412N structures, suggest that the structure and electostatics of the imidazole ring of histidine are critical for its function as a zinc ligand in alkaline phosphatase. |
==About this Structure== | ==About this Structure== | ||
| - | 1HQA is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with ZN as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Alkaline_phosphatase Alkaline phosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.1 3.1.3.1] Full crystallographic information is available from [http:// | + | 1HQA is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=ZN:'>ZN</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Alkaline_phosphatase Alkaline phosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.1 3.1.3.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1HQA OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Kantrowitz, E | + | [[Category: Kantrowitz, E R.]] |
[[Category: Ma, L.]] | [[Category: Ma, L.]] | ||
[[Category: ZN]] | [[Category: ZN]] | ||
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[[Category: transferase]] | [[Category: transferase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:03:46 2008'' |
Revision as of 11:03, 21 February 2008
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ALKALINE PHOSPHATASE (H412Q)
Overview
Site-specific mutagenesis has been used to replace His-412 with glutamine in Escherichia coli alkaline phosphatase. In the wild-type enzyme His-412 is a direct ligand to one of the catalytically important zinc atoms (Zn1) in the active site. The mutant enzyme (H412Q) exhibited about the same k(cat), but a 50-fold increase in K(m) compared to the corresponding kinetic parameters for the wild-type enzyme. Furthermore, the H412Q enzyme had a lower zinc content than the wild-type enzyme. In contrast to the wild-type enzyme, Tris was less effective in the transferase reaction and dramatically inhibited the hydrolysis reaction of the H412Q enzyme. The addition of zinc to the mutant enzyme increased the k(cat) value above that of the wild-type enzyme, partially restored the weak substrate and phosphate binding, and also alleviated the inhibition by Tris. The structure of the H412Q enzyme was also determined by X-ray crystallography. The overall structure of the H412Q enzyme was very similar to that of the wild-type enzyme; the only alpha-carbon displacements over 1 angstrom were observed near the mutation site. In the H412Q structure no phosphate was bound in the active site of the enzyme; however, two water molecules were observed where phosphate normally binds in the wild-type enzyme. Close examination of the active site of the H412Q structure revealed structural changes in Ser-102 as well as at the mutation site. For example, the carbonyl oxygen of the side chain of Gln-412 rotated away from the position of His-412 in the wild-type structure, although too far away (3.2 angstroms) to coordinate to Zn1. Studies on the H412Q enzyme, and a comparison of the H412Q and H412N structures, suggest that the structure and electostatics of the imidazole ring of histidine are critical for its function as a zinc ligand in alkaline phosphatase.
About this Structure
1HQA is a Single protein structure of sequence from Escherichia coli with as ligand. Active as Alkaline phosphatase, with EC number 3.1.3.1 Full crystallographic information is available from OCA.
Reference
Kinetic and X-ray structural studies of a mutant Escherichia coli alkaline phosphatase (His-412-->Gln) at one of the zinc binding sites., Ma L, Kantrowitz ER, Biochemistry. 1996 Feb 20;35(7):2394-402. PMID:8652582
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