Reading for today: Chapter 16 (selections from Sections A, B and C) Friday and Monday: Chapter 17 (Diffusion)

Transcription

1 Lecture 29 Enzymes Reading for today: Chapter 6 (selections from Sections, B and C) Friday and Monday: Chapter 7 (Diffusion) 4/3/6

2 Today s Goals Michaelis-Menten mechanism for simple enzyme reactions: k k 2 E + S ES P + E k - Many of our favorite enzymes are much more complex! But we can use the concepts learned throughout the course to describe the following types of systems: Multiple substrates: Ping-Pong and Sequential enzyme mechanisms llostery in enzymes: we ll use TCase as an example Catalytic mechanisms and resulting kinetic properties: we ll use serine proteases and creatine kinase as examples 4/3/6 2

3 Michaelis-Menten Kinetics Michaelis-Menten equation: v 0 V Lineweaver-Burk plot: v max K M [S] K M V k V K max k k M 0 max Vmax 2 [S] k 2 k cat [E] 0 [E] 0 K M v 0 slope V max [S] K M V max 4/3/6 3

4 Enzyme inhibitors v 0 Competitive displace the substrate through reversible binding to the active site v 0 [S] increase the K M Noncompetitive irreversible substrate analogs or bind away from the active site but interfere with catalysis uncompetitive v 0 [S] [S] reduce V max Uncompetitive Figure from The Molecules of Life ( Garland Science 2008) interact with enzymes in a substrate-dependent manner reduce V max and K M by the same factor 4/3/6 4

5 Enzyme and inhibitor mechanisms Kinetic analysis of an enzyme binding to substrate and two inhibitors gives the results shown in the following graphs. One of the inhibitor is a transition state analog. Identify the inhibitor and explain your reasoning. Inhibitor a Inhibitor b ***If irreversibly bound (which is plausible for a transition state analog), it could also be b. transition state analog binds within the active site and mimics the structure of the transition state between substrate(s) and product(s). Therefore, it should bind competitively with the substrate. These LB plots show a competitive inhibitor in (a) (V max unchanged; K m increased) and a noncompetitive inhibitor in (b) (K m unchanged; V max decreased). Therefore a is the transition state analog. 4/3/6 5

6 Moving beyond simple kinetics Enzymes with multiple substrates E + + B E + P + Q e.g. DN and RN polymerases and the ribosome have both the growing polymer and the incoming monomer Some enzymes bind the substrates in an ordered manner Some enzymes can bind substrates in random order Some enzymes react with one substrate first before they bind the second substrate Ping-pong mechanism 4/3/6 6

7 Bacterial histidine kinase Che uses a ping-pong mechanism Che is a kinase that first transfers the g-phosphate of TP onto its own His residue, then transfers it onto an sp residue in CheY CheY-P Che-His TP Che-His CheY-P Che-His TP Che-His-P CheY Che-His-P DP CheY Che-His-P DP 4/3/6 7

8 Example of ping-pong mechanism Ping-pong mechanism: P B Q E E E*P E* E*B EQ E The first substrate binds E reacts to form the modified enzyme E* and st product P The first product P is released The second substrate B binds E*B react to regenerate enzyme E and form the 2 nd product Q The 2 nd product Q is released, allowing the cycle to start again 4/3/6 8

9 Ping-Pong Kinetics EQ Q E K M E v Vmax K M [B] K [][B] 0 B M [] E*B B K M B E* P E*P v 0 K V M max [] K V B M max [B] What would a Lineweaver-Burk plot look like? What rate might you measure to get v 0? Holding [B], then varying [] allows you to measure the Lineweaver-Burk plot Repeat the experiment, but with 2[B] how is the Lineweaver-Burk plot affected? 4/3/6 9

10 Ping-Pong Kinetics v 0 K V M max [] K V B M max [B] slope K M V max [B] K B M V max [B] v 0 K K B M M [B] [] Intuitive explanation: this is analogous to an uncompetitive inhibitor, except increasing [B] accelerates the reaction rather than slowing it down 4/3/6 0

11 More bimolecular mechanisms Random-order sequential binding mechanism: B E E EB E + B B EB If one substrate is saturating, the mechanism will reduce to a Michaelis- Menten behavior for the non-saturated substrate Ordered sequential binding mechanism: E + E + B EB E + B If [] is saturating, the mechanism and kinetics reduce to Michaelis-Menten behavior 4/3/6

12 K D Sequential kinetics E B K M E EB E + B B K D B EB B K M K M K D K B M [B] K B M v 0 V max [] V max [B] Lineweaver-Burk plot of reactions varying [] and holding [B]? What if you repeat with 2[B]? 4/3/6 2

13 Sequential kinetics K D E E B EB K M E + B K M K D K B M [B] K B M v 0 V max [] V max [B] B K D B EB B K M K B M V max [B] v 0 slope K M K V D K max B M [B] [B] Plot makes intuitive sense: [] Changing [B], we are increasing [EB] The more [B], the more chances to get the reaction forward (increase in rate), so the apparent V max increases 4/3/6 3

14 llostery in enzymes n early step in the synthesis of pyrimidines is performed by aspartate transcarbamylase (TCase): Bond rotations downstream step would be ring closure Figure from The Molecules of Life ( Garland Science 2008) 4/3/6 4

15 TCase is multimeric TCase is a dodecamer: C 6 R 6 6 catalytic subunits arranged as 2 trimers 6 regulatory subunits arranged as 3 dimers Sigmoidal shape indicates Figure from The Molecules of Life ( Garland Science 2008) 4/3/6 5

16 Mechanism of substrate allostery on TCase Thinking back to the example of substrate specificity of HIV protease, what are possible mechanisms of substrate allostery? Binding of PL is cooperative, which demonstrates that allostery is at the level of substrate binding, not chemical reaction rate Figure from The Molecules of Life ( Garland Science 2008) 4/3/6 6

17 Pyrimidines provide negative feedback Figure from The Molecules of Life ( Garland Science 2008) 4/3/6 7

18 Feedback regulation of TCase CTP, one of the downstream products, is a negative regulator Why is TP regulator? 4/3/6 8

19 CTP causes conformational changes 4/3/6 9

20 Peptide hydrolysis Uncatalyzed peptide hydrolysis in water Single-step mechanism DG is small but favorable E a is large slow reaction Relatively non-specific Figure from The Molecules of Life ( Garland Science 2008) 4/3/6 20

21 Catalysis by trypsin Trypsin, like many proteases, has a catalytic triad sp-his-ser + First step of proteolysis is a nucleophilic attack by the serine oxygen 4/3/6 2

22 In a second step, the same His residue polarizes an attacking water molecule - What kind of enzyme mechanism does trypsin use? Figure from The Molecules of Life ( Garland Science 2008) 4/3/6 22

23 Ping-pong mechanism of trypsin P Trypsin-Ser P-P Trypsin-Ser P Trypsin-Ser P-P Trypsin-Ser-P H 2 O Trypsin-Ser-P P H 2 O Trypsin-Ser-P P 4/3/6 23

24 Serine protease catalytic mechanism 4/3/6 24

25 Catalysis by trypsin Trypsin accelerates peptide hydrolysis using several strategies High local concentration proximity of the peptide bond to be hydrolyzed with the attacking serine Favors proper orientation of the substrates Oxyanion hole with peptide amide groups stabilizes the transition states 4/3/6 25

26 Stabilizing the transition state The oxyanion hole has mainchain amide groups pointing towards the carbonyl oxygen Favors the oxygen position of the transition state 4/3/6 26

27 Catalysis by trypsin Trypsin accelerates peptide hydrolysis using several strategies High local concentration proximity of the peptide bond to be hydrolyzed with the attacking serine Favors proper orientation of the substrates Oxyanion hole with peptide amide groups stabilizes the transition states ~0 0 -fold acceleration over aqueous solution k cat ~ 22 s - K M ~ 45x0-6 M k cat /K M ~ 0 5 s - M - 4/3/6 27

28 Proteases have specificity Trypsin cleaves C-terminal of positively charged residues K or R Substrate Cleavage Pn P4 P3 P2 P P P2 P3 P Pm S4 S3 S2 S S S2 S3 S4 Protease Protease Fig. Schematic representation of enzyme-substrate complex with eight binding sites. Positions Pn to Pm in the substrate are counted from the bond between P and P, where the cleavage occurs. ccording to Schechter and Berger (967). P Substrate 4/3/6 28

29 Serine proteases with different specificity Serine proteases share the catalytic mechanism Have distinct specificity Trypsin C-terminal of K,R Subtilisin Broad specificity Tripeptidyl peptidase 3 residues from N-term HIV protease (aspartyl protease) or ngiotensin converting enzyme (CE; zinc-dependent protease) 8-residue specificity Figure from The Molecules of Life ( Garland Science 2008) 4/3/6 29

30 Creatine kinase nother phosphoryl transfer reaction Random order sequential Single-step (no covalent intermediate) Phosphocreatine acts as an energy reservoir, because the DG is small, and the reaction readily reverses when [TP] decrease Figure from The Molecules of Life ( Garland Science 2008) 4/3/6 30

31 Creatine kinase stabilizes transition state nitrate from the crystallization buffer mimics the transition state conformation Knowles, Science 2003 Nitrate is a transition state analog or transition state mimic Figure from The Molecules of Life ( Garland Science 2008) 4/3/6 3

32 Some concepts to remember Many enzyme reactions with more complex mechanisms can be reduced to apparent Michaelis-Menten kinetics Sequential mechanisms Ping-pong Several steps, transition states Enzymes often use multiple strategies to increase the reaction rates Trypsin uses a ping-pong mechanism and several strategies to accelerate peptide hydrolysis 4/3/6 32