Enzymes: Biological Catalysts

Introduction to Enzymes

• Enzymes are proteins that act as biological catalysts.
• Enzymes accelerate biochemical reactions within cells.
• Cells rely on numerous biochemical transformations for survival.
• Each biochemical reaction requires a specific enzyme.
• Enzymes dictate both the rate and direction of biochemical changes.
• The study of enzymes and their properties is crucial for understanding metabolism.

Expected Learning Outcomes

• Define enzymes and explain their basic nature.
• Explain the different naming conventions for enzymes.
• Describe the chemical nature of enzymes and their catalytic efficiency.
• Explain the specificity of enzyme action and regulation of their activity.
• Classify enzymes into six major groups based on biochemical actions.

Nomenclature of Enzymes

• W. Kühne introduced the term "enzyme" in 1878, referring to substances in yeast responsible for fermentation.
• Enzymes are specialized proteins that act as biocatalysts in metabolic reactions.
• Over two thousand enzymes have been identified so far.
• Enzymes facilitate millions of chemical reactions at high speeds under moderate conditions
• Enzymes have medicinal and commercial applications, including measuring enzyme levels in blood to assess heart damage.
• Enzymes have been used in industrial alcohol production for centuries.
• Systematic names are used, common names are derived from the substrate and reaction type, with the suffix "-ase" added
• Lactate dehydrogenase (LDH) converts lactate to pyruvate, removing hydrogen atoms in the process: CH3-CH(OH)-COO- + NAD+ <-> CH3-C(O)-COO- + NADH + H+
• Urease converts urea and water to carbon dioxide and ammonia: H2N-CO-NH2 + H2O -> CO2 + 2NH3
• Alcohol dehydrogenase converts ethanol and NAD+ to ethanal and NADH: CH3CH2OH + NAD+ <-> CH3CHO + NADH
• Initially, nomenclature worked well, but the International Commission on Enzymes (EC) was established by the International Union of Biochemistry (IUB) in 1957.
• The commission recommends systematic and trivial enzyme names, revising the nomenclature over time.
• Known enzymes receive code numbers based on classes and subclasses.
• Code numbers comprise four parts, included in an enzyme list that is followed by common names that are deemed recommended names
• The first number indicates the type of reaction catalyzed.
• The second number indicates the subclass as per the substrates involved.
• The third number indicates the sub-subclass, specifying the reaction.
• The fourth number is the enzyme's serial number within its sub-subclass.
• Lactate dehydrogenase's number is 1.1.1.27 and hexokinase is 2.7.1.10, for example.
• Although systematic names accurately describe enzymes, trivial names are more user-friendly and remain in biochemical literature.

Classification of Enzymes

• The Enzyme Commission (EC) classifies enzymes using EC numbers with four parts.
• Enzymes are named by adding '-ase' to the substrate they act upon.
• Classification is based on the type of reaction an enzyme catalyzes.
• The classification system is based on the reactions enzymes catalyze and the substrates they act on.

Oxidoreductases

• This class of enzymes participates in physiological oxidation-reduction processes and catalyze electron transfer
• For example, alcohol: NAD oxidoreductase uses alcohol as the electron donor and NAD+ as the electron acceptor.
• Ethyl alcohol + NAD+ is converted to acetaldehyde + NADH + H+ by the enzyme
• Also known as alcohol dehydrogenase, but the common name does not fully describe the reactions substrates

Transferases

• This class of enzymes catalyzes the transfer of a chemical group from one substrate to another like amino, methyl, alkyl, or phosphate; the transferred groups could be amino, methyl, alkyl, acyl, sulphate or phosphate, etc.
• For example, hexokinase, systematically named ATP: D-hexose 6-phosphotransferase
• ATP is the phosphate donor, D-hexose is the acceptor, and the transfer happens to the 6-carbon hydroxyl on hexose.
• ATP + D-Hexose becomes ADP + D-Hexose 6-phosphate via hexokinase.

Hydrolases

• This class of enzymes catalyzes hydrolytic reactions.
• Digestive enzymes like amylase, sucrase, lipase and proteases belong to this group.
• These enzymes lead to the breakdown of food materials.
• Pancreatic lipase, degrades lipids and known by its systematic name triacylglycerol acylhydrolase.
• Triacylglycerol + H2O is converted to 2- Monoacyglycerol + 2 Fatty acids via Pancreatic lipase

Lyases

• This class of enzymes facilitates the elimination of chemical groups sans hydrolysis, creating double bonds.
• Fructose bisphosphate aldolase (trivial name) is an example.
• Its systematic name is D-fructose-1, 6-bisphosphate-D-glyceraldehyde 3-phosphate lyase.
• The substrate, D-fructose-1, 6-bisphosphate, degrades to D-glyceraldehyde-3-phosphate; Fructose - bisphosphate aldolase converts D-Fructose -1, 6-bisphosphate to D-Glyceraldehyde 3-phosphate and Dihyroxyacetone phosphate

Isomerases

• Enzymes in this class catalyze isomerizations, including racemases, epimerases, and mutases.
• Triosephosphate isomerase, classified as D-glyceraldehyde 3-phosphate ketol-isomerase, converts D-glyceraldehyde 3-phosphate (aldose) into dihydroxyacetone phosphate (ketose).
• D - Glyceraldehyde 3-phosphate converts to Dihydroxyacetone phosphate via Triosephosphate isomerase

Ligases (Synthetases)

• Enzymes in this class join two molecules, using the energy of a broken high-energy bond.
• Isoleucyl-tRNA synthetase (trivial name) is known as L-isoleucine: tRNAile ligase (AMP-forming) systematically.
• L-isoleucine links to isoleucine-specific tRNA, which splits ATP to produce AMP and pyrophosphate.
• ATP + L-Isoleucine + tRNAile turns into AMP + Pyrophosphate + L- Isoleucyl-tRNAile

Characteristics of Enzymes

• Were considered associated with living organisms and structures, until Buchner demonstrated in 1897 they're organic substances not requiring cellular organization for activity.
• Sumner established the chemical nature of enzymes 30 years later, showing that crystallized urease had protein characteristics
• Urease converts Urea and H2O into 2NH3 and CO2
• Northrop and Kuntiz confirmed this by crystallizing several enzymes and establishing their protein nature.
• In 1960, Stein and Moore determined the amino acid sequence of ribonuclease and Merrifield achieved its total synthesis 9 years later, confirming that enzymes do not differ from other nonbiological origin chemicals; Ribonuclease catalyzes the breakdown of ribonucleic acid

Chemical Nature

• The three-dimensional structure of lysozyme determined by Philips in 1965 allowed his group to present a catalytic mechanism.
• This accomplishment led to the ability to achieve the primary amino acid sequence and three-dimensional structure of many enzymes.
• This has revealed general features that govern enzyme structure, function, regulation, and evolution.
• Enzymes exhibit considerable structural diversity.
• Simple protein molecules, the protein molecule itself, is the only true catalyst for many enzymes.
• Conjugated protein molecules require nonprotein molecules for catalytic function.
• Apoenzyme refers to the protein part
• Cofactor refers to the nonprotein part, which may be metal ions, or complex organic molecules like nicotinamide
• Holoenzyme refers to the combination of an apoenzyme and a cofactor.
• Activator refers to a metal ion cofactor
• Coenzyme refers to a nonprotein organic molecule cofactor
• Prosthetic group describes a tightly bound cofactor
• Substrate describes the catalytic transformation catalyzed by the enzyme
• Active site describes the portion of an enzyme where events of process occur
• Active sites consist of amino acid side chains and binding or catalytic groups.
• Binding amino acids can be either polar or nonpolar, interacting via hydrogen bonds and electrostatic or hydrophobic interactions.
• Polar-type catalytic groups initiate electronic changes as a prelude to reactions.

Catalytic Efficiency

• Enzymes perform under mild temperature and pH conditions in living cells.

• Reactions catalyzed by enzymes hardly occur outside the cell, because it would take nearly 50 years to digest a single meal without them.

• The reversible reaction between carbon dioxide and water to produce carbonic acid is also catalysed by carbonic anhydrase, an enzyme in erythrocytes (Red Blood Corpuscles, RBCs).

• Carbonic anhydrase converts CO2 and H2O to H2CO3

• The rate of this reaction, with the presence of enzymes it is increased to about 107 times.

• Enzymes catalyze reactions by a factor of 10^4 to 10^14 greater than non catalyzed reactions.

• Hydrogen peroxide to oxygen and water is catalyzed by peroxidase

• Peroxidase affects an increase in the rate of reaction by about 1010 as compared to the uncatalysed reaction; Hydrogen peroxide turns into H2O via Peroxidase

• Catalytic efficiency relies on turnover number, that indicates # of substrate molecules transformed in a time unit by an enzyme molecule

• The optimum temperature and pH conditions are also important for turnover number

Specificity of Action

• Specificity distinguishes enzymic from nonenzymic catalysts.
• Enzymes act on a particular substrate for a particular reaction.
• Physiologically specificity refers to the ability of an enzyme to recognize and transform on particular substrates in a mixture
• The degree of specificity varies from enzyme to enzyme
• Enzymes with low specificity act on a variety of substrates containing a susceptible bond, such as a peptide, phosphate ester, or carboxylic ester bond.
• These are degradative enzymes such as peptidases, phosphatases, or esterases.
• Enzymes with intermediate specificity display "group specificities" e.g., carboxypeptidase A removes C-terminal amino acids with a free carboxyl group.
• Hexokinase catalyses phosphorylation of a variety of D-hexoses.
• Enzymes with absolute specificity act on a single substrate for example, urease, which splits only urea.
• Many enzymatic reactions display stereochemical specificity e.g., D-amino acid oxidase acts on D-amino acids only.
• Biosynthetic enzymes tend to be highly specific for anabolic activities
• The “lock and key” hypothesis by Email Fischer said enzymes were highly specific for one substrate like only one particular key opens up to one key.
• Still holds, but the specificity is determined by active site shape, topographical features, and amino acid residues
• Some suggest the 'lock and key' theory for enzyme substrate interaction, where precise active site fits substrate

Induced Fit Theory

• Requires not only a lock and key fit but flexibility for the best fit.
• Constitutes induced fit theory, comparable to hand slipping into a glove to induce the best fit
• Specificity is still retained because a left-handed glove does not fit a right hand.

Regulation of Enzyme Activity

• Ability to be regulated by small ions/molecules sets enzymic catalysis apart from nonenzymic catalysis.
• Regulatory molecules may be substrate, substrate analogues, or structurally unrelated substances.
• Regulatory molecules may be end products, which inhibit pathway-early enzymes.
• This is significant for enabling fine turning of enzyme activity in response to conditions inside a cell