1jxj
From Proteopedia
(New page: 200px<br /> <applet load="1jxj" size="450" color="white" frame="true" align="right" spinBox="true" caption="1jxj, resolution 1.99Å" /> '''Role of mobile loop...) |
|||
| Line 1: | Line 1: | ||
| - | [[Image:1jxj.gif|left|200px]]<br /> | + | [[Image:1jxj.gif|left|200px]]<br /><applet load="1jxj" size="350" color="white" frame="true" align="right" spinBox="true" |
| - | <applet load="1jxj" size=" | + | |
caption="1jxj, resolution 1.99Å" /> | caption="1jxj, resolution 1.99Å" /> | ||
'''Role of mobile loop in the mechanism of human salivary amylase'''<br /> | '''Role of mobile loop in the mechanism of human salivary amylase'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The nonreducing end of the substrate-binding site of human salivary | + | The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase. |
==About this Structure== | ==About this Structure== | ||
| - | 1JXJ 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 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:// | + | 1JXJ 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=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=1JXJ OCA]. |
==Reference== | ==Reference== | ||
| Line 15: | Line 14: | ||
[[Category: Homo sapiens]] | [[Category: Homo sapiens]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Mishra, P | + | [[Category: Mishra, P J.]] |
[[Category: Ragunath, C.]] | [[Category: Ragunath, C.]] | ||
[[Category: Ramasubbu, N.]] | [[Category: Ramasubbu, N.]] | ||
| - | [[Category: Thomas, L | + | [[Category: Thomas, L M.]] |
[[Category: Wang, Z.]] | [[Category: Wang, Z.]] | ||
[[Category: CA]] | [[Category: CA]] | ||
| Line 30: | Line 29: | ||
[[Category: salivary]] | [[Category: salivary]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:27:52 2008'' |
Revision as of 11:27, 21 February 2008
|
Role of mobile loop in the mechanism of human salivary amylase
Overview
The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase.
About this Structure
1JXJ 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
Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity., Ramasubbu N, Ragunath C, Mishra PJ, Thomas LM, Gyemant G, Kandra L, Eur J Biochem. 2004 Jun;271(12):2517-29. PMID:15182367
Page seeded by OCA on Thu Feb 21 13:27:52 2008
Categories: Alpha-amylase | Homo sapiens | Single protein | Mishra, P J. | Ragunath, C. | Ramasubbu, N. | Thomas, L M. | Wang, Z. | CA | CL | Amylase | Catalysis | Enzyme | Human | Mobile loop | Mutagenesis | Salivary
