6.1 A Brief Look at Enzyme Energetics and Enzyme Chemistry Converting substrates to product requires intermediate states – Intermediates are less stable – Can be referred to as transition state Activation energy – Difference in energy level between substrate and transition state – Enzymes lower activation energy

6.1 A Brief Look at Enzyme Energetics and Enzyme Chemistry Most enzymes are proteins Chemical transformation does not change Active site – Surface of the enzyme where substrates bind – Bind in precise orientation with other ancillary groups

6.2 The Enzyme Assay and Initial Velocity Enzyme activity – number of moles of substrate converted to product per unit time Requires an assay Alkaline phosphatase is an example Reaction velocity – ratio of change in concentration to change in time

6.2 The Enzyme Assay and Initial Velocity Enzyme kinetics uses portion of curve illustrated by dashed line

6.3 Simple Kinetic Mechanisms Steady State Assumptions – Necessary to derive an equation form the model that relates initial velocity to substrate concentration – Concentration of enzyme – substrate complex ES is constant with time – Enzyme conservation Can be expressed as the sum of all forms of Enzyme

6.3 Simple Kinetic Mechanisms Michaelis Menton Equation – V i = V max [S]/([K m ] + [S]) – Vmax is maximum velocity of the enzyme – Km – steady – state constant

6.4 How the Michaelis – Menten Equation Describes Enzyme Behavior Michaelis Menton equation predicts the form of this equation Represents steady state with net flow of substrate to product

6.5 The meaning of Km Substrate concentration at ½ Vmax Concentration of substrate at cellular concentration in most cases Different substrates of the same enzyme have different Km – Can use to differentiate between them Low values of Km indicate tighter binding affinity

6.6 – Reversible Inhibition An inhibitor that binds to and dissociates from an enzyme form reaching equilibrium Most common way enzymes are regulated – Cellular regulators – Drugs Bind to active sites of enzymes

6.6 – Reversible Inhibition Can bind different forms of an enzyme – EI produces E and I – ESI produces ES and I Three main types – Competitive – Anticompetitive – Mixed

6.6 Reversible Inhibition Competitive – Binding of the inhibitor to free enzyme form only – Bear chemical structure similarity to the substrate Both I and S bind to the same site on enzyme – Michaelis Menton Equation changes – No change in maximum velocity of enzyme – Effective at low substrate concentrations – Lowers Km/Vm[S] – Slope is decreased

6.6 Reversible Inhibition Anticompetitive – also called Uncompetitive – Binding of inhibitor to ES complex only – Do not resemble substrate – ESI intermediate forms – At point where substrate concentration reaches zero, no effect of the inhibitor – Inhibition is the strongest when [S] reaches infinity

6.6 Reversible Inhibition Mixed – also called noncompetitive – Binding of inhibitor to both E and ES – At low concentration substrate inhibition resembles competitive – At high substrate concentration, inhibition resembles anticompetitive

6.7 The Double Reciprocal or Lineweaver Burk Plot Michealis Menton curve can be transformed into a straight line - Plot 1/v versus 1 / [S] Values of Km and Vmax can be read from the intersections of its axes

6.7 The Double Reciprocal or Lineweaver Burk Plot -- Competitive – lines intersect on Y axis – Anticompetitive – lines are parallel – Noncompetitive – lines intersect on X axis

6.8 Allosteric Enzymes Not all enzymes display velocity – substrate behavior like a Michaelis – Menton enzyme Have modifiers of enzyme bind to a place other than the active site – Modifiers can influence active site remotely – Can cause conformational change of the enzyme Usually multi subunit proteins – Subunits interact during catalysis

6.8 Allosteric Enzymes Displays cooperatively – Catalysis at one subunit increases catalysis at other subunits – Inhibitors shift curve to the right – Activator shift curve to the left Covalent modifications of enzymes can lead to alteration in velocity – Phosphoryl group from ATP is a common one

6.9 Irreversible Inhibition Modifying agent is covalently attached Cannot be removed Leaving enzyme with little or no activity

6.9 Irreversible Inhibition Example – nerve gas diisoproylfluorophosphate – Irreversible inhibitor of acetylcholine esterase – If it can’t work correctly, muscles go into uncontrolled contraction and death results Drug examples – Aspirin – inhibits prostaglandin and prostacyclin formation – Penicillin – disrupts the formation of bacterial cell walls – Prilosec – inhibits proton pump in the stomach

6.11 Enzyme Categories Many kinds of chemical reactions in biochemistry Reactions can be categorized into a few general types Six major classes – Oxidoreductases – Transferases – Hydrolases – Lyases – Isomerases – Ligases

6.11 Enzyme Categories Oxidoreductase – also known as dehydrogenases or reductases – Substrates changes oxidation state in going to product – Mobile cofactor that removes or adds those electrons – More enzymes in this class than any other Transferase – Move a piece of one substrate to another – An example is the transfer of phosphate

6.11 Enzyme Categories Hydrolase – Use to be a subset of transferases – Water is a substrate – Enzymes located in exterior of cells – Cofactors other than water are unnecessary Lyase – Catalyze the splitting of a substrate into pieces – Do not involve water as a substrate

6.11 Enzyme Categories Isomerase – Catalyze rearrangements – Often alters stereochemistry – Other names – racemase and epimerase Ligase – Catalyzes the joining reaction – Involved high energy done – Sometimes called synthetases

6.12 Enzyme like Qualities of Membrane Transport Proteins Embedded proteins in cell membranes enable selective exchange of small molecules Called transporters – Allows specific molecules or class of molecules to cross the membrane Glucose transporters are a great example – Follows Michaelis Menten kinetics when graphed