2018 Biochemistry 110 California Institute of Technology Lecture 11: Enzyme Regulatory Strategies

Transcription

1 2018 Biochemistry 110 California Institute of Technology Lecture 11: Enzyme Regulatory Strategies 1. Aspartate Transcarbamoylase (ATCase) 2. Zymogen and Digestive Enzyme Regulation 3. Blood Clotting and emophilia The Five Standard Regulatory Strategies: 1. Allosteric Control - cooperativity of Multiple active sites Clio and Euterpe or British armony, - feedback inhibition Vol. 1, Isozymes - multiple variations of the same enzyme 3. Covalent Modification Reversible - Ser/Thr Kinases, Tyr Kinases and Phosphatases 4. Proteolytic Activation Irreversible - regulatory cascades 5. Control of Transcription Biochem 111

2 ATCase catalyzes the Committed Step in the Biosynthesis of Pyrimidines This is the ATCase Step: It is effectively irreversible in the Pyrimidine Biosynthesis pathway (committed step). Everything that makes it through this step will eventually end up as C, T or U. It is the optimum opportunity for feedback regulation. These are the subsequent steps:

3 ATCase Enzyme may be Separated into Regulatory and Catalytic Domains Electron Micrograph of ATCase (you can see triangular form in a couple of the ATCase enzymes that are oriented correctly!) (you don t have to squint!) ATCase 100Å diameter, ~300kDa (11.6 Svedbergs) ATCase displays: Sigmoidal kinetics Feedback inhibition by CTP! Multimeric enzyme with cooperative binding of S! allosteric (regulatory) sites on separate polypeptide chains. c 3 catalytic subunit three 34kDa chains (5.8 Svedbergs) ultracentrifugation, etc. r 2 regulatory dimer two 17kDa chains (2.8 Svedbergs) The c 3 catalytic subunit catalyzes reaction, but does not respond to CTP. The r 2 regulatory subunit binds CTP, but does not catalyze reaction. Mixing c 3 and r 2 reconstitutes fully active ATCase: 2c 3 + 3r 2! c 6 r 6

4 The c6r6 Structure of the ATCase Enzyme 20 0Å! ATCase T state (1RAI), side view ^ " top view down three-fold axis of symmetry

5 PALA is a Competitive Inhibitor of ATCase (substrate analog) The Binding of PALA induces a T! R Transition This should remind you of what we learned for emoglobin cooperative binding of 2 : The first S to bind has to turn on the enzyme from T to R then the others can hop on. That makes the enzyme more sensitive to changes in substrate concentration. Allosteric Constant: L = [T]/[R]

6 CTP Effects Feedback Regulation by favoring ATCase in the T state

7 Lactate Dehydrogenase (LD) is a Tetrameric Enzyme derived from two Isozymes = heart ; M = Muscle NAD + NAD C 3 C - 2 Lactate LD C 3 C 2 - Pyruvate 4 M 4 birth increase in serum levels of the 4 relative to the 3 M isozyme is an indicator of a recent heart attack.

8 Protein Modification Strategies

9 Protein Regulation by Phosphorylation/Dephosphorylation Involves Kinases and Phosphatases Serine and Threonine Kinases Share a Common Kinase Domain An active area of Cancer Drug Development - Anti-Angiogenesis Inhibitors (VEGF) - Cell Cycle (JAK) - Cell Development (ALK) C-terminal Lobe inge Region ATP Substrate N-terminal Lobe! substrate: -RRNSIinhibitor: -RRNAI-

10 Proteolytic Activation of Zymogens Releases Active Peptidases The pancrease releases about 1.5 L of enzyme juice each day! These dense (protein shows up dense on EM) zymogen granules are released into the duodenum where a cascade of activation is initiated by enteropeptidase:

11 Blood Clotting is also Accomplished by a Cascade Leading to Fibrin (uch!) emophilia is a sex-linked recessive characteristic leading to Factor X deficiency: "Factor X" serine protease! Thrombosis is the formation of blood clots in blood vessels. Fibrin synthesis requires Vitamin K for the conversion of glutamate to the modified amino acid γ- carboxyglutamate. Anticoagulants are used to treat thrombosis by inhibiting Vitamin K activation.

12 Fibrin Contains the Modified Amino Acid γ-carboxyglutamate Which Requires Vitamin K C 3 6 C 3 6 C 3 2 (text has structural typo here) C 3 ~ N ~ 2, C 3 C 2 6 γ-glutamylcarboxylase C 3 C 3 Vitamin K Vitamin K 2 oxidoreductase Vitamin K dithiol-s 2 dithiol-(s) 2 ~ N ~ C 3 6 dithiol-(s) 2 C 3 Vitamin K C 3 dithiol-s 2 6