Hemoglobin and Myoglobin Myoglobin (Mb) : stores O2 Hemoglobin (Hb) : transports O2 Each of the four chains in Hb looks like Mb, and each carries a heme. Hemoglobin contains two identical a chains and two identical b chains. Letters A–H are a-helical regions. The protein 1) protects heme from oxidation and 2) provides a pocket where only O2-like molecules can fit

The geometry of iron coordination in oxymyoglobin: The octahedral coordination of the iron ion. The iron and the four nitrogens from protoporphyrin IX lie nearly in a plane. A histidine (F8, or His 93) occupies one of the axial positions, and O2 the other. Schematic drawing of the heme pocket, showing the proximal (F8; His93) and distal (E7; His64) histidine side chains.

UV-Vis spectrum of Heme Proteins Changes in the visible spectrum of hemoglobin. Spectra for hemoglobin in the deoxygenated state (blue trace) and the O2-bound state (red trace) are shown. Hemoglobin in the deoxygenated state is a venous purple, whereas completely oxy-Hb is bright red. As more O2 binds to Hb, the visible spectrum shifts from the blue to the red trace

Binding of O2 by Mb & Hb Fe moves 0.3 Ǻ back toward plane of heme

Binding Curves K = equilibrium association constant

Binding Curves are ubiquitous in science & medicine Some Examples… Dose-response curves Ligand (drug) / receptor binding curves Enzyme/substrate activity curves

Oxygen Transport: why Hb works Efficiency in O2 transport is achieved by cooperative binding in multisite proteins, described by a sigmoidal (S-shaped) binding curve. Sigmoidal curves are also seen for DNA melting, enzyme activity)

The conformational change caused by O2 binding Many salt bridges and H-bonds are broken and relocated upon conversion for deoxy to oxy What happens in between? (how does the allosteric change happen?) Above: salt bridges in deoxy Hb. Note the importance of acid-base chemistry

Conformational changes in Hb during the deoxy-oxy transition Oxygenation leads to a 15° rotation of one αβ dimer with respect to the other. Note also how the inner pocket changes shape.

Bohr Effect How does this work? For example, in muscle tissue, the pH is lower (more acidic) than in the lungs. The higher [H+] helps to drive O2 unloading in the capillaries.

Binding of BPG lowers affinity of Hb for O2

Sickle-cell anemia Normal Hb  normal erythrocyle Hb S  sickled erythrocyte hydrophobic pimple from Glu  Val mutation at β6 Hb S forms a long fibrous polymer of linked Hb S One aa change  molecular disease…REMARKABLE! Sickle-cell anemia confined to people originating in tropical area because people with α2ββ(s) are more resistant to malaria

Enzymes: Transition State Theory Transition State (T.S.) : high energy species that is formed transiently en route from reactants to products Free energy of reaction (ΔGo): difference in free energies of reactants and products. Free energy of activation(ΔG‡): energy difference between reactants and the top of the hill, i.e. the highest energy T.S. ΔG‡ is also called the activation energy (Ea).

How do enzymes work? Ea determines the kinetics of a reaction. If Ea is large  reaction is slow If Ea is small  reaction is fast Enzymes speed up reactions by lowering the activation energy (the height of the hill) Note: the thermodynamics (ΔG0) of the reaction are unchanged – the enzyme only speeds up the reaction

Enzyme – Substrate Complexes Substrate: reactant in the reaction catalyzed by the enzyme Enzymes bind substrates in a specific location, called the active site, to form enzyme substrate complexes (ES complexes)

Stereospecificity conferred by an enzyme: Active sites have 3-D structure Chiral substrates bind specifically to the active site (ex. Epinephrine) The asymmetric surface of an enzyme can even confer stereospecific binding with a symmetric substrate.

2 Models for Enzyme-Substrate Interactions Lock and Key: active site of enzyme is complementary in shape to the substrate Enzyme + Substrate = ES A static model

2 Models for Enzyme-Substrate Interactions Induced Fit: Active site not rigid Shape modified through binding of substrate Shape of active site complementary to that of the substrate only after substrate is bound Enzyme + Substrate = ES A model of dynamic recognition

Introduction to Enzymes Enzymes are very selective about which substances they interact with and which reaction they catalyze. This specificity, along with the regulation of enzymatic activity, is what allows the many diverse metabolic pathways to be harmoniously interconnected for life.

Michealis Menten Kinetics Enzyme activity (or velocity V) can be modulated by: substrate concentrations phosphorylation allosteric effectors (like hemoglobin)

Example: Hexokinase The binding of glucose to hexokinase induces a significant conformational change in the enzyme. The enzyme is a single polypeptide chain, with two major domains. Notice how the obvious cleft between the domains (panel a) closes around the glucose molecule. Copyright © 2013 Pearson Canada Inc.

Serine Proteases

Chymotrypsin structure The structure of chymotrypsin and the serine protease catalytic triad: The backbone of bovine chymotrypsin detemined by X-ray crystallopgraphy. Here, H57 is protonated, S195 and D102 are deprotonated. Copyright © 2013 Pearson Canada Inc.

Chymotrypsin mechanism (1) Catalysis of peptide bond hydrolysis by chymotrypsin: Copyright © 2013 Pearson Canada Inc.

Chymotrypsin mechanism (2) Catalysis of peptide bond hydrolysis by chymotrypsin: Copyright © 2013 Pearson Canada Inc.

Linear Plots: Lineweaver-Burk

Linear Plots: Eadie-Hofstee Graphing v versus V/[S], we obtain Vmax at (V/[S]) = 0 and KM from the slope of the line.

Enzyme Inhibition: Competitive

Enzyme Inhibition: Noncompetitive

Fast Reactions Stopped-flow Pushing triggers data collection begins. Mixing takes ~ 1 ms, so limited to enzymes with kcat < 103 s-1 31

Temperature Jump For really fast kinetic processes (sub-millisecond processes) Say reaction mixture is at equilibrium at T1 … Quickly increase T1 to T2 by passing current between electrodes in the mixture, then watch relaxation to new equilibrium ∆ [A](t) = (∆ A)tot e-t/ = relaxation time  gives rate constants 1/  = k1 + k-1 For 32

Contributions of various His residues to the Bohr effect Copyright © 2013 Pearson Canada Inc.