Enzymes and Amino Acids

• Enzymes are proteins with catalytic properties due to their specific activation power.
• Enzymes are common in biochemical systems of biosensors and are the first to demonstrate the potential for commercialization of biosensors.
• Enzymes are biopolymers of α-amino acid residues linked by amide bonds.
• There are 20 amino acids; all are crucial as building blocks of proteins.
• Cysteine forms covalent disulfide bridges in proteins or with a thiolated solid support like gold (Au-S bonds) in oxygenated environments and is frequently involved in enzyme or biosensor functions.
• Enzymes act as reaction catalysts.

Enzyme Properties

• The majority of enzymes function at temperatures below 50 °C.
• The physiological pH range for most enzymes is pH 5-8.

Amino Acids

• Acidic amino acids include aspartate and glutamate.
• Basic amino acids include arginine, lysine, and histidine.
• pH changes affect protein structure through interactions like hydrogen bonds, disulfide bonds, electrostatic forces, and van der Waals forces.

Protein Structure

• Primary structure is the sequence of amino acids.
• The 3D structure of the protein is determined by the primary structure.
• Secondary structures include alpha-helices and beta-sheets.
• Tertiary structures are more complex.
• Quaternary structures involve multiple subunits.

Enzyme Structure and Reconstruction

• Enzymes are proteins with a globular shape and a complex 3-D structure.
• Human pancreatic amylase is an example of an enzyme.
• Enzymes have a protein part(globular)
• Co-factors are non-protein chemical compounds that act as helper molecules.
• Coenzymes receive redox equivalents, protons, and chemical groups from the substrate during the enzymatic reaction.

Cofactors

• Tightly bound cofactors are called prosthetic groups.
• Cofactors that are easily bound and released are called coenzymes.
• Many vitamins serve as coenzymes.
• Vitamin C acts as a cofactor in biosynthetic and gene regulatory enzymes which are involved in the synthesis of collagen, carnitine, and catecholamine hormones.
• Nicotinamide adenine dinucleotide (NADH) is a common cofactor involved in the function of 250 enzymes.

Substrates and Active Sites

• The substrate of an enzyme is the reactant that is activated by the enzyme.
• Enzymes are specific to their substrates.
• Specificity is determined by the active site.
• Active sites are a crucial part of enzymes.
• The shape and chemical environment within the active site enables reactions.
• Amino acid residues in active sites are not always close in the primary sequence but come together in a 3D structure.
• Activity is affected by structure.

Chemical Reactions and Catalysis

• Chemical reactions need an initial input of energy: activation energy.
• During a reaction, molecules pass through a transition state.
• Increasing temperature makes molecules move faster, but biological systems are temperature-sensitive.
• Enzymes increase reactions without raising temperature by lowering activation energy.
• Enzymes create a new reaction pathway or "short cut".
• Enzyme-controlled reactions are 108 to 1018 times faster than non-enzymatic reactions.
• Urease accelerates the hydrolysis of urea by a factor of about 10^14.

Allosteric Site

• An allosteric site exists where an effector molecule binds to alter the catalytic activity of an enzyme.
• Allosteric site is different from the active site.
• Allosteric effectors are small, non-protein molecules.
• Products of the enzyme-substrate reaction can act like allosteric inhibitors.
• Not all enzymes have allosteric sites, but all are sensitive to effectors that stabilize the signal in the reaction media.

Lock and Key Hypothesis

• The fit between the substrate and the active site is exact.
• It's analogous to a key fitting into a lock.
• The substrate is like the key, and the enzyme like the lock.
• A temporary enzyme-substrate complex is formed.
• Upon completion, products with a different shape are released, freeing the enzyme for another substrate.
• Lock and key hypothesis explains enzyme specificity and the loss of activity when enzymes denature.

Induced Fit Hypothesis

• Some proteins can change their shape (conformation).
• An enzyme induces a conformational change when a substrate combines.
• The active site is molded into a precise conformation.
• Chemical environment becomes suitable for easier reaction.
• Substrate bonds stretch, lowering activation energy.
• This explains that enzymes can react with a range of similar substrates.

Enzyme Specificity

• Include linkage, absolute, group and stereochemical specificity.

Linkage Specificity

• The enzyme recognizes a specific type of bond in different molecules, independent of the rest of the molecular structure

Absolute Specificity

• Enzymes can recognize only one type of substrate and implement their catalytic functions.

Group Specificity

• The enzyme acts on a specific functional group within a molecule, regardless of the rest of the structure

Stereochemical Specifity

• The enzyme can act on only one form of isomers of the substrates.

Factors Affecting Enzymes

• Substrate concentration
• pH
• Temperature
• Inhibitors

Substrate Concentration

• In non-enzymatic reactions, the increase in velocity is proportional to the substrate concentration.
• In enzymatic reactions, the reaction rate increases until it reaches a saturation point when all enzyme molecules are occupied.
• If the concentration of the enzyme is altered, then the Vmax will change.

Effect of pH

• Extreme pH levels cause denaturation.
• Enzyme structure changes are due to pH.
• The active site is distorted, and the substrate molecules can no longer fit.
• Changes in the charge of the enzyme and its substrate occur at pH values that are slightly different from the enzyme's optimum value.
• Ionization changes affect the binding of the substrate with the active site.

Effect of Temperature

• Q10 (temperature coefficient) is the increase in reaction rate with a 10°C rise in temperature.
• For chemical reactions, Q10 = 2 to 3: the reaction rate doubles or triples with every 10°C rise.
• Enzyme-controlled reactions follow this rule as they are chemical reactions.
• Proteins denature at high temperatures.
• The optimum temperature for an enzyme-controlled reaction balances Q10 and denaturation.
• Most enzymes have an optimum temperature of about 30°C.
• Some enzymes function at lower temperatures; cold water fish will die at 30°C because their enzymes denature.
• Very few bacteria enzymes can withstand very high temperatures up to 100°C.
• Most enzymes are fully denatured at 70°C.

Inhibitors

• Inhibitors are chemicals that reduce the rate of enzymic reactions.
• They are usually specific and work at low concentrations.
• Inhibitors block enzymes, but they do not usually destroy them.
• Many drugs and poisons are inhibitors of enzymes in the nervous system.

The Effect of Enzyme Inhibition

• Irreversible inhibitors combine irreversibly with the functional groups of the amino acids in the active site.
• Reversible inhibitors can be washed out by dialysis
• Nerve gases and pesticides containing organophosphorus combine with serine residues.

Competitive Reversible Inhibitors

• They compete with the substrate molecules for the active site.
• The inhibitor's action is proportional to its concentration.
• The inhibitor resembles the substrate's structure closely.
• An example of this involves succinate dehydrogenase

Non- Competitive Reversible Inhibitors

• Non-competitive inhibitors are not influenced by the concentration of the substrate
• They inhibits by binding reversibly to the enzyme but not at the active site.
• Cyanide combines with iron in cytochrome oxidase.
• Heavy metals, Ag or Hg, combine with -SH groups.
• The non-competitive inhibitors can be removed by using a chelating agent such as EDTA.

Enzyme Classes

• Transferases, Hydrolases, Lyases, Isomerases, Ligases

Enzyme Biosensor Applications

• Used to analyze concentration, identification, enzyme kinetics, inhibitor concentration (toxic species, chemical warfare agents)
• Used to label in affinity sensors.