1bsi
From Proteopedia
(New page: 200px<br /> <applet load="1bsi" size="450" color="white" frame="true" align="right" spinBox="true" caption="1bsi, resolution 2.Å" /> '''HUMAN PANCREATIC ALPH...) |
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| - | [[Image:1bsi.gif|left|200px]]<br /> | + | [[Image:1bsi.gif|left|200px]]<br /><applet load="1bsi" size="350" color="white" frame="true" align="right" spinBox="true" |
| - | <applet load="1bsi" size=" | + | |
caption="1bsi, resolution 2.Å" /> | caption="1bsi, resolution 2.Å" /> | ||
'''HUMAN PANCREATIC ALPHA-AMYLASE FROM PICHIA PASTORIS, GLYCOSYLATED PROTEIN'''<br /> | '''HUMAN PANCREATIC ALPHA-AMYLASE FROM PICHIA PASTORIS, GLYCOSYLATED PROTEIN'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic | + | Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile. |
==About this Structure== | ==About this Structure== | ||
| - | 1BSI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with NAG, CA and CL as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http:// | + | 1BSI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=NAG:'>NAG</scene>, <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=CL:'>CL</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1BSI OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Homo sapiens]] | [[Category: Homo sapiens]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Brayer, G | + | [[Category: Brayer, G D.]] |
| - | [[Category: Cote, H | + | [[Category: Cote, H C.F.]] |
[[Category: Hewitt, J.]] | [[Category: Hewitt, J.]] | ||
| - | [[Category: Macgillivray, R | + | [[Category: Macgillivray, R T.A.]] |
[[Category: Numao, S.]] | [[Category: Numao, S.]] | ||
| - | [[Category: Overall, C | + | [[Category: Overall, C M.]] |
| - | [[Category: Rydberg, E | + | [[Category: Rydberg, E H.]] |
[[Category: Sidhu, G.]] | [[Category: Sidhu, G.]] | ||
| - | [[Category: Vo, H | + | [[Category: Vo, H C.]] |
[[Category: Wang, Y.]] | [[Category: Wang, Y.]] | ||
| - | [[Category: Withers, S | + | [[Category: Withers, S G.]] |
[[Category: CA]] | [[Category: CA]] | ||
[[Category: CL]] | [[Category: CL]] | ||
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[[Category: pichia pastoris]] | [[Category: pichia pastoris]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:58:40 2008'' |
Revision as of 09:58, 21 February 2008
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HUMAN PANCREATIC ALPHA-AMYLASE FROM PICHIA PASTORIS, GLYCOSYLATED PROTEIN
Overview
Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.
About this Structure
1BSI is a Single protein structure of sequence from Homo sapiens with , and as ligands. Active as Alpha-amylase, with EC number 3.2.1.1 Full crystallographic information is available from OCA.
Reference
Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris., Rydberg EH, Sidhu G, Vo HC, Hewitt J, Cote HC, Wang Y, Numao S, MacGillivray RT, Overall CM, Brayer GD, Withers SG, Protein Sci. 1999 Mar;8(3):635-43. PMID:10091666
Page seeded by OCA on Thu Feb 21 11:58:40 2008
Categories: Alpha-amylase | Homo sapiens | Single protein | Brayer, G D. | Cote, H C.F. | Hewitt, J. | Macgillivray, R T.A. | Numao, S. | Overall, C M. | Rydberg, E H. | Sidhu, G. | Vo, H C. | Wang, Y. | Withers, S G. | CA | CL | NAG | Amylase | Catalysis | Diabetes | Enzyme | Glycosylated protein | Human | Hydrolase | Mutagenesis | Pancreatic | Pichia pastoris
