References on Kinetics and Mechanisms

References on Kinetics and Mechanisms Excellent reference for all aspects of enzyme kinetics including important elements of Metabolic Control Analysis of relevance to systems analysis of enzyme function and regulation: Fundamentals of Enzyme Kinetics 4 th Edition (2012) Athel Cornish-Bowden, Wiley-Blackwell, ISBN 978-3-527-33074-4 (great historical notes and excellent plot visuals as bonuses) Straightforward presentation of theory, methods, examples for enzyme kinetics, catalysis and folding: Structure and Mechanism in Protein Science (1999) A. Fersht, Freeman. Extensive descriptions of enzyme mechanisms with overall and active site structures of the enzymes: Enzymatic Reaction Mechanisms (2007) PA Frey & AD Hegeman, Oxford Univ. Press. In depth reference for biologically relevant kinetics Kinetics for the Life Sciences: Receptors, Transmitters, and Catalysts (1995) H. Gutfreund, Cambridge University Press Older encyclopedia of SS kinetics eqns for multiple substrates, all types of product and reversible inhibition Enzyme Kinetics (1975) I.H. Segel, Wiley & Sons.

Last Week Kinetics time-dependence of processes Rates, macroscopic rate constants Reaction models, microscopic rate constants Free energy profiles Enzymes Rates, steady-state kinetics, energy profiles Structural contributions to catalysis

This Week Enzymes Structural contributions to catalysis - Recap, compare and contrast designed and natural enzyme Enzymes with complex regulation and other functions (Ras/Ras-GAP/Ras-GEF/Kinases) Kinetics Analyzing individual steps in an enzyme reaction - Ras kinetics on Wed

Long standing concepts [TS ] in the TS Noncovalent binding interactions maximized at TS E+A obs ΔG bind A EA additional favorable ΔG bind felt here and Q EQ unfavorable ΔG destab felt only in GS E+Q Environmental changes that make A more reactive than in solution

Recap Kemp Eliminase - C-H proton abstraction - Anionic product - 1-step irreversible b/c structural change

Recap Kemp Eliminase - Snug fit - Hydrophobic & dehydrated - Precisely aligned Asp-COO - - Well ordered Gln orients GS, stabilizes TS

Compare KE with TIM reaction J. R. Knowles 1970s, 80s, 90s, many others 90s - current

TIM Key Structural Features Contributing to Catalysis Similar features a) Compact, hydrophobic, desolvated b) 2 main catalytic residues: Glu, His Additional features c) Loop closure (i) positions Glu (base) (ii) locks in substrate, (iii) dehydrates active site cavity with hydrophobics d) Pi substrate-assisted closure and specificity

Compact reactive parts of substrate buried TIM: DHAP yellow C, PDB: 1NEY

TIM Catalytic residues Fig. from J. Richard, (2012) Biochemistry 21, 2652. 1.2 Å structure from Jogl, et al (2003) PNAS 100, 50. Glu165 B: His95 polarizes C=O Lys12 binds P i and may help polarize DHAP C=O

TIM Atom motions within the box a) Overlay of DHAP - cyan enediolate analog - magenta 1.2 Å structure from Jogl, et al (2003) PNAS 100, 50. d) Anisotropic motions of atoms in active site only in directions productive for catalysis

TIM the lid to the box that has many critical features! Loop 6 closure essential for proper placement of Glu165! Malabanan, et al (2010) Curr Opin Struct Biol 20, 702.

TIM the lid to the box that has many critical features! Pi substrate-assisted closure essential for dehydration, catalyst placement in hydrophobic tunnel specificity Malabanan, et al (2010) Curr Opin Struct Biol 20, 702.

TIM the lid to the box that has many critical features! Loop hinge is rigid! - Hinge mutations cause enhanced nanosecond motions of loop - large loss of entropy on closure results in 2500-fold lower k cat. YE-PGG-AIGTG-GGG-TP Sun & Sampson (1998) Protein Sci 7, 1495.

TIM the lid to the box that has many critical features! Ile172 in Loop 6 pairs with Leu232 to form a hydrophobic cage around Glu165 restricts it to motions perpendicular to C1-C2 bond of substrate, and likely increases its pka so a better base catalyst to remove H + from C. Malabanan, et al (2010) Curr Opin Struct Biol 20, 702.

What else contributes to catalysis? Interactions at a distance

Control of Cellular Pathways Ras and Ras-like GTPase Domains Timer Switches With Auxiliary Controllers

GPCRs Trimeric G-Proteins Ras-like Gα GTPase Domain Receptor 7-transmembrane helical domain Directly binds Ras-like Gα Results in exchange of GDP for GTP Kobilka (2011) Nature 477, 549

Regulation of Growth Factor Signaling Pathways - Ras GTPases Growth factors bind to receptor-tyrosine kinases causing receptor dimerization which leads to autophosphorylation on Tyr of cytosolic domains Images from Molecular Cell Biology 4 th Ed. Lodish, Berk, Zipursky et al. NY: W.H. Freeman, 2000.

Regulation of Growth Factor Signaling Pathways Growth factors bind to receptor-tyrosine kinases causing receptor dimerization which leads to autophosphorylation on Tyr of cytosolic domains GRB2 adaptor binds Tyr-OPi Sos binds adaptor and Ras-GDP Images from Molecular Cell Biology 4 th Ed. Lodish, Berk, Zipursky et al. NY: W.H. Freeman, 2000.

Regulation of Growth Factor Signaling Pathways Growth factors bind to receptor-tyrosine kinases causing receptor dimerization which leads to autophosphorylation on Tyr of cytosolic domains GRB2 adaptor binds Tyr-OPi Sos binds adaptor and Ras-GDP GDP Sos catalyzes exchange of GDP for GTP yielding activated Ras-GTP complex GTP Images from Molecular Cell Biology 4 th Ed. Lodish, Berk, Zipursky et al. NY: W.H. Freeman, 2000.

Regulation of Growth Factor Signaling Pathways Activated Ras-GTP binds to effector Ser/Thr protein kinases that switch on cell growth pathways Fig. 4 from Pino & Chung (2010) Gastroenterology 138, 2059.

In both Ras and Ras-like Gα domains, hydrolysis of GTP to GDP leads eliminates binding to effectors Ras requires activation of hydrolysis by GAP, Gα hydrolyzes GTP much faster than Ras but can also be stimulated Ras-GDP GEFs Guanine nucleotide Exchange Factors, e.g. Sos GAPs GTPase Activating Proteins, e.g. p120gap Ras-GTP INACTIVE conformation - cannot bind effectors ACTIVE conformation can bind effectors

Ras-like Gα-GTPγS complex vs Ras-GDPCP complex Kobilka (2011) Nature 477, 549 PDB: 6Q21 reported in Milburn et al (1990) Science 247, 939

Hydrolysis causes conformational changes in Switch I & II regions Active Ras-GDPCP complex Switch II Inactive Ras-GDP complex Switch I PDB: 6Q21 reported in Milburn et al (1990) Science 247, 939 PDB: 1Q21 reported in Milburn et al (1990) Science 247, 939

Hydrolysis causes conformational changes in Switch I & II regions Active Ras-GDPCP complex (GDP-CH 2 -P analog) Inactive Ras-GDP complex guanine ribose Mg 2+ P loop Switch I extra CH 2 PO 3 2- Switch II PDB: 6Q21 reported in Milburn et al (1990) Science 247, 939 PDB: 1Q21 reported in Milburn et al (1990) Science 247, 939

Hydrolysis causes conformational changes in Switch I & II regions Active Ras-GTP complex (GDP-CH 2 -P analog) Inactive Ras-GDP complex Switch I Switch II PDB: 6Q21 reported in Milburn et al (1990) Science 247, 939 PDB: 1Q21 reported in Milburn et al (1990) Science 247, 939

Molecular Interactions in Ras-GTP Complex Milburn et al., (1990) Science 247, 939 - Oncogenic mutations frequently at Q61 & G12 not stimulated by GAP - A59T autophosphorylates and is oncogenic

Regulation of Growth Factor Signaling Pathways Pacold et al. (2000) Cell 103, 931. Switch 1 and Switch 2 regions of Ras-GTP (below) bind to effector proteins e.g. PI 3-Kinase γ (right) Milburn et al (1990) Science 247, 939.

Complex of Ras-GTP with PI3Kγ Pacold et al. (2000) Cell 103, 931.

Regulation of Growth Factor Signaling Pathways Pacold et al. (2000) Cell 103, 931. Switch I

Regulation of Growth Factor Signaling Pathways Hydrolysis of GTP to GDP leads to conformational change in Switch 1 & Switch 2 regions eliminating binding to effectors Ras-GDP GEFs Guanine nucleotide Exchange Factors, e.g. Sos GAPs GTPase Activating Proteins, e.g. p120gap Ras-GTP INACTIVE conformation - cannot bind effectors ACTIVE conformation can bind effectors

Complex of Ras-GTP with PI3Kγ Pacold et al. (2000) Cell 103, 931.

Regulation of Growth Factor Signaling Pathways Pacold et al. (2000) Cell 103, 931. Switch I

Regulation of Growth Factor Signaling Pathways Mutations that block GTP hydrolysis are common in cancer Ras-GDP GEFs Guanine nucleotide Exchange Factors, e.g. Sos GAPs GTPase Activating Proteins, e.g. p120gap Ras-GTP INACTIVE conformation - cannot bind effectors ACTIVE conformation can bind effectors

Regulation of Growth Factor Signaling Pathways Regulation of Enzyme Kinetics of Small GTPases Critical for Proper Regulation of Cellular Signalling Pathways P i Ras+GTP k 1 Ras GTP k 2 Ras GDP P i k 3 Ras GDP k 4 Ras+GDP k -1 k -2 k -3 P i k -4 1) Which steps are slow? Paper for Wed 2) Which steps are enhanced by protein-protein interactions with GAPs and GEFs and how? Paper for Wed looks at kinetics of Ras-Gap, for Fri looks at Ras-GAP structure 3) How is control coupled to protein dynamics? J Gross next week