Download Amylase v1

Diversity in the Structure and Function of Amylase
Kim Gernert and Vedham Karpakakunjaram
Target audience:
Students enrolled in Principles of Biology I (BI 107) and II (BI 108)
Background
Alpha amylase: http://www.rcsb.org/pdb/101/motm.do?momID=74
Alpha-amylase begins the process of starch digestion. It takes starch chains
and breaks them into smaller pieces with two or three glucose units.
Human salivary amylase is used in one of our lab modules, so students are
familiar with the enzyme and its function.
Questions
• Given organisms from three domains with diverse lifestyles and study
sequence differences and their effect on enzyme’s structure and function.
• Are the structures of amylase different across organisms?
• Relate the identity and percentage similarities in sequences based on
clustering in the phylogenetic tree.
• Relate the conserved regions of the alignment to the secondary structure
of amylase.
• Identify the active site and locate the critical residues and find their
conservation of the sequences in the alignment.
• Study the binding of other molecules in the active site including inhibitors.
• How do mutations in amylase modify their structures?
• How do these modifications alter the enzyme’s function?
Homo_sapie
ALGKDYVRSKIAEYMNHLID-IGVAGFRIDASKHMWPGDIK--------------AILDK
n3VM5_Meda
ALEKDYVRGKVADFMNKLID-MGVAGFRVDACKHMWPGDLD--------------NVYRR
Tenebrio_m
NQGSDYVRGVLIDYMNHMID-LGVAGFRVDAAKHMSPGDLS--------------VIFSG
sp|P25718|
GTFHGGDLRGLTNKLDYLQQ-LGVNALWISAPFEQIHGWVGGGTKGDFPHYAYHGYYTQD
Saccharomy
RTEDSDVASVFNSWVKDFVGNYSIDGLRIDSAKHVDQGFFP-------------DFVSAS
Oryza_sati
DHLNKRVQRELIGWLDWLKMDIGFDAWRLDFAKGYS----------------------AD
Pyrococcus
THELVYERGWLKEFFDRISSDDKINLMLYSEYLSKFRPKGLVYLPIASYFEMSEWSLPAR
. .. : . .
Homo_sapie
LHNLNSNW-FPEGSKPFIYQEVID----LGGEPIKSSDYFGNGRVTEFKYGAK—LGTVI
n3VM5_Meda
LNNLNTKW-FPGGSRPFIFQEVID----LGGEPITTGEYVGLGRVTEFKYGAR—LGELF
Tenebrio_m
LKNLNTDYGFADGARPFIYQEVID----LGGEAISKNEYTGFGCVLEFQFGVS—LGNAF
sp|P25718|
WTNLDANMGNEADLRTLVDSAHQRGIRILFDVVMNHTGYATLADMQEYQFGALYLSGDEV
Saccharomy
GVYSVGEVFQGDPAYTCPYQNYIP----------GVSNYPLYYPTTRFFKTTDSSSSELT
Oryza_sati
MAKIYIDATEPSFAVAEIWTSMAN----------GGDGKPNYDQNAHRQELVNWVDRVGG
Pyrococcus
QAKLFFEFIKKLKELNLFEKYRIFVRG------GIWKNFLYKYPEGNYMHKRMLMLSKLL
Tools:
• Amino acid sequences (PDB, EBI) from many diverse organisms to be
provided for students to select about 5-6 organisms representing the three
domains.
 Hyperthermophilic Archeae, parasitic and mutualistic bacteria,
unicellular and multicellular eukaryotes.
• Compare the sequences with alignments from CLUSTALW and construct
phylograms in: www.phylogeny.fr
• Use NCBI (http://www.ncbi.nlm.nih.gov/) for comparing the identity and
percent similarities in the sequences across organisms in order to
synthesize the information from phylogenetic trees.
• Use Chimera (www.cgl.ucsf.edu/chimera) to visualize:
 conserved and modified regions, especially around the active site.
 the binding of substrate, inhibitors
 to determine the nature of inhibition (competitive)
 Provide sequences of mutations to be compared against normal sequences for
identification of conserved and modified regions, and to predict modifications in
the mutant enzyme’s function, if any.