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Slide 1:

Chymotrypsin is made in cells in an inactive form, secreted from cells. Must be cleaved to fully activate. (chymotrypsin is a “ zymogen ”) Why is chymotrypsin made in an inactive form? (can’t have an active protease loose inside cells)

Slide 2:

Ritonavir is a protease inhibitor (Abbott laboratories). Believed to work by inhibiting proteases that degrade other HIV inhibitors, allowing other HIV inhibitors to persist longer in body. Ritonivir is used in low doses as part of a multi-drug cocktail. Enzyme inhibitors - May look like substrates, and compete with natural substrate for active site.

Slide 3:

Saquinavir is an inhibitor of the HIV protease. The natural substrate of HIV is a Phe - Pro peptide bond. Saquinavir mimics the natural substrate of HIV protease, and is a competitive inhihitor of HIV protease. Saquinavir was the result of “rational drug design” also known as “structure-based drug design”.

Slide 4:

Chymotrypsin is an example of an “ acid-base catalyst ”. What’s that? Mechanism involves a.a. acting as acids and bases. In chymotrypsin mechanism, Serine donates proton, His accepts proton.

Slide 5:

Amino acid side chains that can act as acid-base catalysts What do these have in common? What side chains can NOT be acid-base catalysts?

Slide 6:

“ covalent catalysts ” Chymotrypsin is also a covalent catalyst. During the mechanism, an intermediate forms a covalent link to the enzyme. Tetrahedral intermediate

Slide 7:

“ Metal ion catalysts ” use a metal ion, bound in their active site, as part of their mechanism.

Slide 8:

K M is the substrate concentration when the initial reaction velocity is one-half of its maximum value. Three numbers that can describe an enzyme’s catalytic abilities are: K M and V max and k cat k cat = number of catalytic cycles by each active site per second.

Slide 9:

Suppose you have a new enzyme, and want to characterize how good of a catalyst it is. What to do? A common procedure is to use the Michaelis-Menten analysis: Measure initial reaction velocity ( v o ) at different substrate concentrations [S] .

Slide 10:

Alternatively, you might find K M and V max from the slope and intercept in a Lineweaver-Burk plot, then calculate k cat . k cat = V max / [E] total

Slide 11:

Lineweaver-Burk plot for an enzyme catalyzed reaction is shown. What is K M for this enzyme? A) -2 uM B) 1 uM C) 1 uM D) 2 uM E) 0.5 uM

Slide 12:

What is V MAX for this enzyme? With units? 1/ V MAX = 2 sec/mM V MAX = 0.5 mM/sec

Slide 13:

1. Regulation by enzyme inhibitors. a) Irreversible inhibitors. b) Competitive inhibitors. c) Mixed inhibitors. d) Allosteric inhibitors. 2. Cells can also regulate enzymes by controlling the amount of enzyme produced. Enzyme activity in cells is regulated. How ?

Slide 14:

An “ irreversible inhibitor ” binds so tightly to the enzyme active site that the enzyme is permanently blocked.

Slide 15:

5-fluorouracil is an irreversible inhibitor of thymidylate synthase. 5-fluorouracil forms a covalent bond with a cysteine in the thymidylate synthase active site, inactivating the enzyme. 5-fluorouracil is used in treating cancer.

Slide 16:

Competitive inhibitors compete with the substrate for an enzyme’s active site.

Slide 17:

A competitive inhibitor alters K M but not V max (This same Vmax makes sense to me, because the inhibitor can be overwhelmed by excess substrate).

Slide 19:

“ Mixed inhibition ”: The inhibitor binds to both the enzyme and ES complex, causing a conformational change that makes the active site less efficient.

Slide 20:

A mixed inhibitor typically alters both V max and K M .

Slide 21:

Allosteric Inhibition. This term is used to describe the regulation of enzymes with multiple active sites : “ Allosteric inhibition ” - A molecule binding to the enzyme causes a conformational change that decreases the catalytic ability of the enzyme. Also, “ allosteric activation ” is observed in some enzymes. A molecule binding to the enzyme causes a conformational change that increases the catalytic ability of the enzyme.

Slide 22:

Example - Glycolysis is regulated by feedback inhibition. Feedback Inhibition . (see next page)

Slide 23:

Feedback inhibition regulates the glycolysis pathway. 10 steps in glycolysis, each catalyzed by its own enzyme. Phosphofructokinase catalyzes step 3. Phosphoenolpyruvate (PEP) is the product of step 9, and it inhibits the enzyme at step 3. Product of step 9 inhibiting the enzyme of an earlier step (step 3) is an example of feedback inhibition. phospho-fructokinase

Slide 24:

Regulation by Phosphorylation. Enzymes called “ kinases ” covalently attach phosphoryl groups to serine, tyrosine, threonine. Phosphorylation of Ser, Tyr or Thr can be an on-switch or off-switch for enzymes.

Slide 25:

Kinases transfer a phosphoryl group (usually from ATP) to an enzyme. Phosphoryl groups are removed by “ phosphatase ” enzymes.

Slide 26:

A phosphatase with Cys and Asp in its active site removes a phosphoryl group from a tyrosine. phosphatase The phosphatase is regenerated, and phosphoryl group is released. substrate Tyr

Slide 27:

Are all proteins enzymes ? Are all enzymes proteins ?

Slide 28:

Until about 1980, all known enzymes were proteins. Then it was discovered that some RNAs can catalyze reactions. These catalytic RNA enzymes are called “ ribozymes ”. One of the first known RNA enzymes is the “ hammerhead ribozyme ”. It is an RNA from a plant virus that can cleave other RNAs. active site

Slide 29:

The ribosome is a large enzyme: It contains over 50 proteins and 3 RNAs, and has a molecular weight of over 2 million. The catalytic center is all RNA, no protein. Translation , the linking together of amino acids to form proteins, is catalyzed by the ribosome.

Slide 30:

RNA of large subunit is gray, RNA of small subunit is cyan, proteins are purple and blue. tRNAs are held between the 2 subunits, and are red & yellow. The active site of the ribosome is all RNA, no protein ! Structure of the ribosome is now known (from x-ray crystallography). Yusupov, Yusupova, Baucom, Lieberman, Earnest, Cate, Noller (2001) Science, 292, 883-896.

Slide 31:

Protein “L9” I spent 3 years working on the structure of protein “ L9 ”, which is one protein in the ribosome. I worked on my little protein from 1990 to 1993. The structure of the whole ribosome was completed in 2000.

Slide 32:

I spent 3 years working on this one protein structure.

Slide 33:

The ribosome structure was completed in 2000, about 7 years after I worked on my small part of it.

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