Enzyme Mechanism - Examples
Determination of Enzyme Mechanisms - A General Approach
1. Purify the enzyme to homogeneity and determine its amino acid sequence and 3-D structure. Compare these basic structural properties of the enzyme to other know amino acid sequences using the computer databases like GenBank maintained by the National Center for Biotechnology Information at NIH (using computer tools like 'ENTREZ' or 'BLAST' - go to HTTP://www.ncbi.nlm.nih.gov/ on the WEB to learn about this database), like Swiss-Prot in Switzerland which has a collection of protein sequences in its database (go to http://expasy.hcuge.ch/sprot/sprot-top.html) and to other protein 3-D structures using 'The Protein Data Base' maintained by Brookhaven National Laboratory (to learn more about protein 3-D structures go to the PDB via HTTP://pdb.pdb.bnl.gov/) or compare your 3-D structure to the PDB using a computer in Germany (go to DALI at http://www.embl-heidelberg.de/dali/dali.html). Finding amino acid sequences and 3-D structures related to the enzyme you are studying will be very helpful in identifying invariant amino acid residues important in the enzyme's structure and functionality.
2. Study the kinetics and substrate specificity of the enzyme and identify inhibitors.
3. Identify key functional amino acid side chains and do 'site-directed mutagenesis' using the cloned gene to confirm the importance of these key amino acid residues. Are they essential for catalytic activity? Are they important for substrate binding? Are they important for stability of the folded native state of the enzyme?
4. Make hypothesis of the chemical events and bond rearrangements occurring during catalysis. Test this hypothesis by 'site-directed mutagenesis' and methods to identify 'intermediates' in catalysis.
These two lectures will illustrate the results obtained in the studies of Enzyme Mechanisms by giving examples for specific enzymes, including an exploration of 'The Active Site' of some specific enzymes. The lectures are divided into 5 topic areas as shown below:
A. pH Effects in Active Sites
Why is chymotrypsin most active at pH 8?
Why is ribonuclease more active at pH 6?
B. Metal Ion Assisted Catalysis
How do Zn-Proteases catalyze peptide bond hydrolysis?
C. Electron Transfers to/from NADH/NADPH
How is stereospecifity achieved in dehydrogenases?
Can an enzyme's disulfide bond be redox reactive?
D. Beta Barrel Enzymes
How does the 'perfect enzyme' work?
How does the most abundant enzyme on earth work?
E. An Enzyme from a Thermophilic Organism
How do enzymes remain stable and function at high temperature?
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu