Altering any molecule requires energy Molecules are stable; must absorb energy to break bonds in a reaction activation energy gets the rxn going energy Need a spark to start a fire cellulose CO2 + H2O + heat

Activation Energy Energy for destabilizing covalent bonds of reactant(s) Moves the reaction over an energy barrier/’hill’ Even if the reaction is exergonic and free energy is liberated glucose 2nd Law of thermodynamics Universe tends to disorder so why don’t proteins, carbohydrates & other biomolecules breakdown? at temperatures typical of the cell, molecules don’t make it over the hump of activation energy but, a cell must be metabolically active heat would speed reactions, but… would denature proteins & kill cells

Reducing Activation energy Catalysts reduce the amount of activation energy needed In cells, catalysts include enzymes (most common) and ribozymes (RNA design; first bio catalyst?) uncatalyzed reaction watch the vocab: catabolic rxn catalyst to initiate rxn Catalase is an enzyme that breaks down H2O2 catalyzed reaction reactant product

Virtually all cell processes require a catalyst replication

Enzymes Vocabulary -ase • enzyme suffix • often reflects molecule name Substrate • reactant which binds to enzyme • enzyme-substrate complex: temporary association Active site • enzyme’s catalytic site; substrate fits into active site Product • end result of reaction active site products substrate enzyme

Properties of enzymes Reaction specific specific enzyme-substrate pairing physical/chemical fit between active site & substrate Not consumed in reaction single enzyme molecule can catalyze thousands or more reactions per second Affected by cellular conditions any condition that affects protein structure A denatured enzyme will not perform function i.e. temperature, pH, salinity

Enzyme models Catabolic: stressing bonds Anabolic: arranging molecules in optimal position to form bonds

Enzyme models • ‘Lock and Key’ ‘Induced Fit’ Perfect 3-D at active site ‘Induced Fit’ Rough fit, substrate binds, enzyme changes shape H bonding between substrate & enzyme

Regulating enzyme function: Cofactors Cofactors activate/enhance enzyme activity any substance required for the proper functioning of an enzyme, such as minerals, ions, vitamins (coenzymes) ex: coenzyme A, NAD (niacin; B3), FAD (riboflavin; B2) Hemoglobin is aided by Fe Chlorophyll is aided by Mg All sequenced genomes encode enzymes that employ coenzyme A in fatty acid & pyruvate metabolism

Regulating enzyme function: Inhibitors Inhibitors prevent the formation of the enzyme-substrate complex, shutting done the pathway a way to end a reaction when no longer needed overcome inhibition by increasing substrate concentration: saturate the solution with substrate so it out-competes inhibitor for active site Competitive inhibition Methanol (wood alcohol) poisoning occurs because methanol is oxidized to formaldehyde and formic acid which attack the optic nerve causing blindness. Ethanol is given as an antidote for methanol poisoning because ethanol competitively inhibits the oxidation of methanol. Ethanol is oxidized in preference to methanol and consequently, the oxidation of methanol is slowed down so that the toxic by-products do not have a chance to accumulate.

examples EX: penicillin blocks active site of the enzyme bacteria use to build cell walls EX: Ethanol  acetaldehyde  acetic acid w/ dehydrogenase w/ aldehyde oxidase 1. Disulfiram (Antabuse) inhibits aldehyde oxidase 2. Acetaldehyde accumulates 3. Side-effects of build up: Methanol (wood alcohol) poisoning occurs because methanol is oxidized to formaldehyde and formic acid which attack the optic nerve causing blindness. Ethanol is given as an antidote for methanol poisoning because ethanol competitively inhibits the oxidation of methanol. Ethanol is oxidized in preference to methanol and consequently, the oxidation of methanol is slowed down so that the toxic by-products do not have a chance to accumulate. Penicillin binds at the active site of the transpeptidase enzyme that cross-links the peptidoglycan strands. It does this by mimicking the D-alanyl-D-alanine residues that would normally bind to this site. Penicillin irreversibly inhibits the enzyme transpeptidase by reacting with a serine residue in the transpeptidase. This reaction is irreversible and so the growth of the bacterial cell wall is inhibited.

Allosteric Sites • Secondary binding sites on an enzyme - an engaged molecule causes a change in shape of active site - can activate or inhibit - alters reaction rate The sites form weak, noncovalent bonds with these molecules, causing a change in the conformation of the enzyme. This change in conformation translates to the active site, which non-competitive inhibition

example • This case is negative feedback poisoning For example, the amino acid alanine noncompetitively inhibits the enzyme pyruvate kinase. Alanine is one product of a series of enzyme-catalyzed reactions, the first step of which is catalyzed by pyruvate kinase. Basis of most chemotherapytreatments is enzyme inhibition. Many health disorders can be controlled, in principle, by inhibiting selected enzymes. Two examples include methotrexate and FdUMP, common anticancer drugs which inhibit enzymes involved in the synthesis of thymidine and hence DNA. Since many enzymes contain sulfhydral (-SH), alcohol, or acid groups as part of their active sites, any chemical which can react with them acts as a noncompetitive inhibitor. Heavy metals such as silver (Ag+), mercury (Hg2+), lead ( Pb2+) have strong affinities for -SH groups. Cyanide combines with the copper prosthetic groups of the enzyme cytochrome C oxidase, thus inhibiting respiration which causes an organism to run out of ATP (energy) Oxalic and citric acid inhibit blood clotting by forming complexes with calcium ions necessary for the enzyme metal ion activator. • This case is negative feedback - product is the inhibitor, shuts down pathway

Metabolic Pathways Reactant to Product requires multiple steps a principal chemical is modified by a series of chemical reactions enzymes catalyze each reaction, often requiring cofactors in order to function properly metabolic pathways can be elaborate!

A metabolic pathway begins with a specific reactant… Enzyme 1 Enzyme 2 Enzyme 3 C D B product reactant A …which is altered, in stages, with the help of enzymes… …producing a final product

Example glycolysis - glucose oxidation to ATP urea cycle - disposal of NH4+ in less toxic forms gluconeogenesis - glucose synthesis from smaller precursors, to be used by the brain.

Factors affecting enzyme function : Inhibitors Ask: Why is the competitive inhibitor less effective?

Concentration: substrate or enzyme X saturated Ask: How is the rate limited? By # of enzymes What if the number of enzymes was far lower than substrate – would concentration of substrate matter?

Temperature -20C 4C 10C 30 30C 60C % substrate remaining

Temperature reaction rate temperature Cold Molecules move more slowly Collisions between enzyme/substrate decreased Heat Molecules energized More frequent collisions Too high = enzyme denatured shape is changed, no longer functions However, increasing temperature also increases the Vibrational Energy that molecules have, specifically in this case enzyme molecules, which puts strain on the bonds that hold them together. As temperature increases, more bonds, especially the weaker Hydrogen and Ionic bonds, will break as a result of this strain. Breaking bonds within the enzyme will cause the Active Site to change shape. This change in shape means that the Active Site is less Complementary to the shape of the Substrate, so that it is less likely to catalyse the reaction. Eventually, the enzyme will become Denatured and will no longer function. temperature

Temperature reaction rate temperature Homo sapiens Thermus thermophilius 37°C 70°C reaction rate temperature (158°F)

pH Changing pH (H+) disrupts the proteins bonds, altering 3D shape

Salinity In high salt, one disrupts the electrostatic interactions because the salt competes for the electrostatic interactions within the protein weakening them. Some salts also disrupt the structure of water which weakens the interactions pushing the protein into its shape. In zero salt R chains will attract to one another, denaturing the protein (enzyme) An intermediate salt concentration such as that of human blood (0.9% ) or cytoplasm ins the optimum for many enzymes.

pH Not all enzymes have the same optimal pH