Enzymes and Bioenergetics

• Enzymes can be globular proteins or RNA.
• Ribozymes are catalytically active RNA.
• Enzymes reduce activation energy and speed up the time to reach equilibrium.
• Enzymes do not influence the equilibrium itself.

Terminology

• ∆G represents free reaction enthalpy.
• ∆H represents reaction enthalpy.
• T is absolute temperature.
• ∆S is reaction entropy.
• ∆G‡ is the activation energy, or the energy content of the transition stage.
• Enzymes can couple exergonic and endergonic reactions.
• Globular proteins are composed of a substrate binding center and a catalytic center, which together form the active center.
• A monofunctional enzyme has one active center.
• A bifunctional enzyme has two active centers (e.g., PFK-B in glycolysis).
• A trifunctional enzyme has three active centers (e.g., LCAT in fatty acid oxidation).

Cofactors

• Enzymes require cofactors.
• Cofactors can be essential ions.
• Activator ions are loosely bound.
• Metal ions of metalloenzymes are tightly bound.
• Cosubstrates are loosely bound.
• Prosthetic groups are tightly bound.

Co-Factors Function

• FMN and FAD (Riboflavin Vit. B2) and NAD+ contribute to proton/electron transfer (Niacin Vit. B3).
• Thiamine pyrophosphate contributes to decarboxylation (Thiamine Vit. B1).
• Pyridoxale phosphate contributes to transamination and decarboxylation (Pyridoxine Vit. B6).
• Coenzyme A contributes to acyl transfer (Panthotenic acid Vit. B5).
• Biotin contributes to carboxylation (Vit. H).
• Tetrahydrofolate contributes to C1-group transfer (Folic acid Vit. B9 = Vit B11).
• Ascorbate contributes to hydroxylation (Vit. C).
• Cobalamine contributes to alkyl-group transfer/methylation (Vit. B12).
• Enzymes exhibit stereoselectivity, substrate specificity, and reaction specificity.

Specificity Notes

• Tyr-tRNA-Synthetase catalyzation: Tyr + ATP + t-RNA Û Tyr-t-RNA + AMP + PPi
• Tyr is 104 times tighter bound than Phe.
• Enzymes inherently possess chirality; contain L-amino acids and an asymmetric active center
• Trypsin only cleaves L-amino acids from polypeptides and not D-amino acids.
• Glutamate + NAD+ becomes a-Ketoglutarate + NH4+ + NADH + H+ via Glutamatedehydrogenase.
• Glutamate + Pyruvate becomes a-Ketoglutarate + Alanine via Alanin-Aminotransferase (ALAT).
• Enzymes often exclude water and they change conformation during catalysis.
• Aspartate + Carbamoylphosphate yields Carbamoylaspartate + Pi via Aspartate-Transcarbamoylase.
• Transition-state analogs bind to enzyme active sites, blocking normal substrate conversion.
• Transition State Analogs are high-affinity competitive inhibitors such as Ritonavir

Catalysis Types

• Acid-base catalysis transfers a proton between the enzyme and substrate using amino acids like aspartate or lysine
• Covalent catalysis forms a covalent bond between the enzyme and substrate, using a nucleophilic functional group in a two-part catalytic process.
• Metal ion catalysis involves catalysts participating in oxidation-reduction reactions via changes in the oxidation state with a cofactor to properly orient the substrate

Enzyme Classes

• Oxidoreductases catalyze oxidation-reduction reactions
• Transferases transfer functional groups
• Hydrolases catalyze hydrolysis reactions
• Lyases cleave C-C, C-O, C-N, and C-S bonds
• Isomerases catalyze isomerization reactions
• Ligases catalyse the formation of bonds
• Orthologs are one enzyme in different species with evolutionary relationships with each other (human, mouse, frog)
• Analogs are enzymes with similar function, but are not evolutionarily related
• Isoenzymes are paralogs

Isoenzymes Characteristics:

• Isoenzymes adapt to different metabolic situations.
• Serve tissue-specific roles
• Express different ontogenetic differences
• Originate from gene duplication.
• Exhibit differences in kinetics, affinity, expression, and regulation.
• Two examples of moonlighting enzymes include Aconitase and Phosphoglucose isomerase (PGI).
• Aconitase is an enzyme that converts Citrate into Isocitrate and translation-regulating factor of mRNAs when there are iron deficiencies
• Phosphoglucose isomerase (PGI) enzyme convert Glucose-6-P into Fructose-6-P and is a fetal life: nerve growth factor
• Enzymes can be regulated by enzyme amount and enzyme activity
• 1 katal equals 1 mol/s, and 1 U equals 1 µmol/min

Factors that enzyme are dependent upon:

• pH
• Temperature
• Substrate concentration
• inhibitors
• Allosteric Modulators
• Covalent Modification
• Protein-Protein Interaction
• Specific enzyme activity is measured in 1 U/mg Protein.
• Key to understand catalytic dynamics through Quantitative descriptions
• Find appropriate inhibitors to influence activity .
• Serves as a clinical indicator
• LDH, ALAT, ASAT, CK, AChE, Clotting factors etc are key factors

Observations on Enzyme Kinetics:

• Enzymes can affect very low concentrations ~ [enzyme] = nM
• Initial rate (velocity) is linear with [enzyme].
• Initial velocity increases with [substrate] at low [substrate]
• Maximum initial velocity at high [substrate].
• The Michaelis-Menten mechanism follows these steps:
  • E + S reversibly forms ES
  • ES proceeds to E + P
• Velocity = v = k2[ES]
• the enzyme binds non-covalently to the substrate to form a non-covalent ES complex

Enzyme Notes:

• A Michaelis complexes is stabilized by molecular interactions and is readily formed or dissociated .
• The enzyme can be free ([E]) or bound ([ES]): [E｡] = [ES] + [E]
• At sufficiently high [S] all enzyme is up as ES (i.e., [E｡] ≈ [ES])
• At high [S] the enzyme is working at full capacity with vmax
• The fill capacity velocity is determined by all kcat and [E｡]
• kcat is the turnover number: number of moles of substrate produced per time per enzyme active site
• KM is the substrate concentration required to reach half-maximal velocity

Enzymes:

• Velocity is expressed with $v = \frac{Vmax \cdot S}{KM + S}$
• kcat describes an enzyme's turnover rate
• Catalyze essentially irreversible reactions and rate limiting
• Regulated by allosteric activators or inhibitors
• Be up or downregulated by allosteric factors at constant [S]
• Don't Need structural resemblance to to any substrae
• Sigmoidal curve when graphed between velocity and (S)

###Transformation Equations V= $\frac{Vmax \cdot S}{(KM + S)}$

• Reciprocal Form: $\frac{1}{Vmax}$ + $\frac{Kn}{Vmax \cdot S}$
• KM = $\frac{Vmax \cdot S}{V-S}$
• Vmax = $\frac{V(KM + S)}{S}$
• The enzyme is either free ([E]) or bound ([ES]): [Eo] = [ES] + [E].
• Specific activity of enzyme = $\frac{umol}{min}$
• Regulation of enzyme activity: competitive and non-competitive, and irreversible Inhibitors
• Covalent Modifications: Irreversible (Limited Proteolysis and Reversible with Interkonversion)
• Phosphorylation GTPase

Enzyme Inhibitors:

-Covalent Binding of Inhibitors -Competitive -Non competative