Lecture 3 Enzymes Ning Flashcards
Enzymes
Catalyze thermodynamically possible biological reactions. Not changed by reaction or used up, doesn’t change reaction position or equilibrium, act by forming a transient complex with reactant, stabilizing transition state. Have optimum temp and pH.
Enzymes vs inorganic catalysts
Accelerate reactions in higher degree, higher specificity, more sensitive to temp and pH (enzymes made of proteins).
Most enzymes are optimum at
37 degC, pH 7. Enzymes will denature above 45-50 degC
Ex of prosthetic group
metalloenzymes have firmly bound metal ions at active site (copper, zinc, iron, cobalt). Vitamins are critical
Holoenzymes/complex
Protein and nonprotein parts
Apoenzyme
Protein part of holoenzyme
Cofactor
Nonprotein part of holoenzyme
Prosthetic group
Small inorganic molecule or atom usually. Tightly bound to apoenzyme typically.
Coenzyme
Large organic molecule loosely bound to apoenzyme. Act as group transfer reagents. Hydrogen, electrons or groups of atoms can be transferred. Metabolite coenzymes or vitamin-derived coenzymes.
Metabolite coenzymes
Synthesized from common metabolites.
Vitamin-derived coenzymes
Derivatives of vitamins.
Vitamins
Cannot be synthesized by animals, must be obtained via diet.
TPP
derivative of thiamine (vit B12). Participates in reactions of Oxidative decarboxylation, Transketo-lase enzyme reactions don’t worry about structure Coenzyme example
Pyridoxal Phosphate
PLP is derived from Vit B6 family of vitamins
PLP is a coenzyme for enzymes catalyzing reactions involving amino acid metabolism (isomerizations, decarboxylations, transamination). All of these - helping transfer a specific group from one molecule to another.
Coenzyme example
Oxidoreductases
Ared + Box→Aox + Bred, enzyme: Dehydrogenases, peroxidases, oxidases
Catalyze oxidation/reduction reactions.
Transferases
A-B + C→A + B-C, enzyme - Hexokinase, transaminases
Catalyze group transfer reactions - some amino acids formed this way.
Hydrolases
A-B + H2O→A-H + B-OH, enzyme = Alkaline phosphatase, trypsin
Catalyze hydrolysis reactions where water is the accepter of the transferred group.
esterases peptidases glycosidases
Pyrophosphate to 2 phosphate groups via phosphatase.
4. Lyases (synthases)
X-A-B-Y→A = B + XY, enzyme = fumarase, dehydratases
Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination
5. Isomerases
A ⇌ isoA, enzyme =Triose phosphate isomerase, phosphogluco-mutase
Catalyze isomerization reactions
Ligases (synthetases)
A + B + ATP→A-B + ADP + Pi enzyme = Pyruvate carboxylase, DNA ligases
Catalyze ligation, or joining of two substrates
Require chemical energy (e.g. ATP)
Glutamine synthetase is an example.
Common names
-ase to end of substrate, don’t describe chemistry of reaction
Trivial names
Don’t give info about enzyme, substrate or product
Enzyme classification number
EC: 2.3.4.2 First digit refers to a class of enzyme, second -to a subclass, third – to a subsubclass, and fourth means the ordinal number of enzyme in subsubclass
Active site
Specific region in the enzyme to which substrate molecule is bound (substrate is usually pretty small).
Characteristics of active site
Specificity (absolute, relative (group), stereospecificity), small 3D region of protein (only 3-5 AAs interact), Binds substrates through multiple weak interactions (noncovalent bonds), there are contact and catalytic regions in the active site.
Fischer theory
The enzyme active site (lock) is able to accept only a specific type of substrate (key)
Absolute Specificity
One enzyme one substrate
Relative specificity
one enzyme acts on different substrates which have the same bond type (example: pepsin splits different proteins)
Stereospecificity
Some enzymes can catalyze the transformation only substrates which are in certain geometrical configuration, cis- or trans. Don’t worry about when an enzyme is absolute or relative. Just another way to describe it.
Enzymatic activity def.
One international unit (IU) of enzyme catalyzes conversion of 1 µmol of substrate to product per minute
The specific activity of an enzyme is a measure of the number of IU/mg protein
Rate of catalysis
At a fixed enzyme concentration [E], the initial velocity Vo is almost linearly proportional to substrate concentration [S] when [S] is small but is nearly independent of [S] when [S] is large. Rate rises linearly as [S] increases and then levels off at high [S] (saturated)
ES complex
Intermediate enzyme-substrate complex
Km
Michaelis constant - Km = 1/2Vmax. When Km is small, increase in substrate results in low increase in rate.
Vo
initial velocity caused by substrate concentration, [S];
=Vmax[S]/(Km+[S])
Vmax
Max velocity - plateau at top of graph
Higher E concentration in fixed saturated S
greater initial reaction rate
Enzyme inhibitors
metabolites, substrate analogs, toxins, drugs, metal complexes, binds to an enzyme and prevents the formation of ES complex or breakdown of it to E + P
Reversible inhibitors
Three basic types of reversible inhibition: Competitive, Uncompetitive, Noncompetitive – after combining with enzyme (EI complex is formed) can rapidly dissociate
Enzyme is inactive only when bound to inhibitor
EI complex is held together by weak, noncovalent interaction
Competitive inhibition
•Inhibitor has a structure similar to the substrate thus can bind to the same active site, higher concentration of substrate can kick it out.
Ex of comp. inhibition
Benzamidine competes with arginine for binding to trypsin
Noncompetitive inhibition
- Binds to an enzyme site different from the active site
- Inhibitor and substrate can bind enzyme at the same time
*Can help reaction, not just hurt it.
•Cannot be overcome by increasing the substrate concentration
Uncompetitive inhibition
Uncompetitive inhibitors bind to ES not to free E
This type of inhibition usually only occurs in multisubstrate reactions
Irreversible inhibitors
very slow dissociation of EI complex
Tightly bound through covalent or noncovalent interactions
3 types: group-specific reagents
- substrate analogs
- suicide inhibitors
Group specific reagents
–react with specific R groups of amino acids
DIPF is an example.
Substrate analogs
structurally similar to the substrate for the enzyme
-covalently modify active site residues
Suicide inhibitors
Inhibitor binds as a substrate and is initially processed by the normal catalytic mechanism
•It then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification
•Suicide because enzyme participates in its own irreversible inhibition (makes product that kills it).
Regulation of enzyme activity
4 main methods: allosteric control, reversible covalent modification, isozymes, proteolytic activation.
Allosteric enzymes
have a second regulatory site distinct from active site. Have quarternary structure. Noncompetitively inhibited enzymes are this.
Allosteric modulator
Allosteric modulators bind noncovalently to allosteric site and regulate enzyme activity via conformational changes. Two types of modulators–positive and negative.
Negative modulator
Bind to active site and inhibit enzyme action. Usually end product feeds back to stop reaction.
End-product inhibition
exactly what it sounds like.
Positive modulator
Binds to allosteric site and stimulates activity, usually rxn substrate.
Isoenzymes/isozymes
Multiple forms of an enzyme which differ in amino acid sequence but catalyze the same reaction. kinetics,
regulatory properties,
the form of coenzyme they prefer and
distribution in cell and tissues
Isoenzymes are coded by different genes
Lactate dehydrogenase is a key example.
Isoenzymes are important for diagnosis of different diseases - acute myocardial infarction (large amount of LDH in heart, not elsewhere - opposite for hepatitis in liver).
Lactate dehydrogenase
5 Isozymes of LDH:
Lactate to pyruvate (NAD+ goes to NADH)
H4 – heart -highest affinity best in aerobic environment
HM3
H2M2
H3M
M4 – liver, muscle -lowest affinity best in anaerobic environment
Zymogens
Inactive precursors to enzymes - activated by proteolytic cleavage.
Proteolytic cleavage
Proteolytic activation only occurs once in the life of an enzyme molecule, Examples of specific proteolysis
•Digestive enzymes
–Synthesized as zymogens in stomach and pancreas
•Blood clotting enzymes
–Cascade of proteolytic activations
•Protein hormones
–Proinsulin to insulin by removal of a peptide
Multienzyme complexes
different enzymes that catalyze sequential reactions in the same pathway are bound together. Usually created when one enzyme that cuts others is cut.
Multifunctional enzymes
different activities may be found on a single, multifunctional polypeptide chain
Metabolite channeling
“channeling” of reactants between active sites. Occurs when the product of one reaction is transferred directly to the next active site without entering the bulk solvent. Can greatly increase rate of a reactions
Channeling is possible in multienzyme complexes and multifunctional enzymes
pH sensitivity of enzymes results from
The effect of pH on the ionic charge of amino acid side chains of enzymes.
Pepsin
Stomach, optimum pH is around 2
metalloenzymes
have firmly bound metal ions at active site (copper, zinc, iron, cobalt).
Group transfer reagents
transfer hydrogens, electrons, groups of atoms
Lactate dehydrogenase
Oxidizes lactate to pyruvate, reduces NAD+ to NADH.
Pyruvate decarboxylases
Lyase - converts pyruvate + H+ to acetaldehyde and CO2
Lysozyme
Active area has 6 AA residues which are far apart in sequence.
Active site functional groups
-OH, -NH, -COO to name a few. Bind through weak interactions (noncovalent).
Salt bridge
- to + interaction
Hydrophobic pocket
Exactly what it sounds like
Small hydrophobic pocket
Exactly what it sounds like - built around target size.
Hexokinase, Glucokinase
Add phosphate group to glucose. Glucokinase is much faster, Km is much larger.
Penicillin
Competitive inhibition. Enzyme that links bacterial cell walls caught up with penicillin. Not a bacteriocidal drug, just prevents bacterial growth.
Phosphorylation
Covalently add phosphate group to enzyme, you can make it more or less active.
Proenzyme
Enzyme activated by cuts. Can lead to cascade by activating other enzymes.
