Biochem 5-7 Flashcards
Enzyme
- Class of proteins that catalyze biochemical reactions
- in many cases extremely improved rates
- stereospecificity
oxidoreductase
oxi-reduct reactions
- often called dehydrogenases
- usually need a coenzyme like NAD+ or NADP+
Catalyst
increase the rate that equilibrium is reached by lowering Ea
Transferases
- catalyze fxnal group transfers
- may require coenzymes
- includes kinases (they transfer phosphates)
Hydrolase
- special kind of transferase that transfers to group to water
- Use water to break apart a compound
Lyase
- Catalyze lysis of the substrate, creating a double bond via elimination rxn
- splits a molecule in two, creating a double bond
Isomerase
- catalyze structural change within a single molecule
- ie. change L alanine to D alanine
Ligase
- catalyze ligation, or joining of two molecules
- also called SYNTHETASES (usually)
- ie change glutamate to glutamine by adding a NH4 molecule
Enzyme Kinetics
- unlike simple kinetics, enzyme kinetics is dependant on the formation of the enzyme-substrate complex. (ES)
- happens in two steps with a distinct rate for each
- depends on S and E
- but if S»»>E than only E matters
initial velocity
E + S –k1 and k-1– ES –k2– E + P
k-2 is negligible
form of ES is rapid and E + P is slower
rate measured at beginning is called initial velocity
Steady State assumption
- ES is formed at the same rate that is decomposes (no net change in ES)
- can be used to derive Michaelis Menten
Catalytic Constant
when S»_space;>E than the rate of the reaction is vmax and rate only depends on E
-vmax = kcat[E]tot
-kcat is the catalytic constant also called the turnover number
simple reactions = 2
Michaelis-Menten Eqn
enzyme catalysis is 1st order
vo = Vmax[S] / Km + [S]
Michaelis constant
Km = Vmax/2
enzyme is half saturated when Km = [S]
Line-weaver Burk
line form of Michaelis Menten
y int = 1/vmax
x int = -1/km
used to calculate Km and Vmax from experimental data
Inhibitor
compound that binds to an enzyme and interferes with its activity
-reversible ones bind via noncovalent forces
-irreversible ones bind covalently
THREE kinds of reversible:
competitive
uncompetitive
noncompetitive
Classical/Competitive Inhib
- most common
- only binds free enzyme (not ES)
- competes with substrate but not always at the same active site
- doesn’t affect Vmax
- raises Km
Nonclassical/Uncompetitive Inhib
- Only bind ES, not the free enzyme
- only in multisubstrate reactions
- both Vmax and Km are decreased.
- parallel lineweaver burk
- includes Allosteric
Noncompetitive Inhib
- Bind to E or ES, effectively removing enzyme molecules from the reaction
- typically not substrate analogs dont bind the same site as the substrate
- decreases Vmax, no change to Km
Irreversible Enzyme Inhib
- form stable covalent adducts with enzymes, effectively killing them.
- often through acylation or alkylation of the active site residues
Enzyme Inhib Example: Organophosphorous Inhibs
- Inactivate hydrolase enzymes through reaction with their serine residues in their active sites
- sarin gas
Affinity Labels
- Irreversible inhibs with affinity for an enzyme’s active site are called affinity labels
- allow for site specific covalent mods
- useful to know which residues are critical for activity
Allosteric enzymes
- bind to another site away from the active site, but cause a change in the active site
- causes conformational change and inhibition
- Do not exhibit standard MM kinetics
- useful for regulation–activators and inhibs
- allosteric modulators noncovalent and arent chem modded by enzyme
- multisubunit enzymes
- sigmodial rate versus [S] for at least one substrate
Regulation by Covalent Mods
- covalent mods to polypeptide chain
- slower than allosteric
- reversible processes, usually catalyzed by other enzymes
- Phosphorylation of hydroxylated or basic AA residues
Two kinds of nucleophilic sub
- tetrahedral intermediate (can be isolated)
2. pentavalent transition state (happens briefly)
Types of enzymatic mechanisms
Cleavage reactions
Oxi-Red reactions
Reaction coordination
- how enzymes can increase the rate of reaction by decreasing Ea
- accomplished by stabilizing transition states
Enzymatic Catalysis
- reactants bound by enzyme brings them higher in free energy (lessens Ea)
- reactants and transition state bound by enzyme lowers transition energy (brings Ea down even further)
Polar residues in active sites
-usually there are polar residues in actvie sites
-reactive fxnal groups
pKa values differ from free AAs
Acid-Base Catalysis
- Proton transfer catalyzes reaction
- often talked about in terms of general acids and bases
Covalent Catalysis
-substrates covalently bonded to enzyme forming a reactive intermediate
-20 % of enzymes use this
-nucleophilic catalysis more common
A net equation would look like this:
AX —> AB with an E intermediate in there thats not part of overall conversion
pH dependence of enzymes
- many have an optimal pH range
- ionizible AAs are the main players in an enzymes active site
Diffusion controlled reactions
-If an enzyme is especially good, than its upper limit is controlled by rate of diffusion
10^8- 10^9 M-1 s-1
Diffusion controlled: Triose Phosphate Isomerase
Interconversion of DHAP and G3P
using glutamate and histidine that shuttle protons
Diffusion controlled: Superoxide Dismutase
degradation of superoxide
-has a Cu atom wit catalytic activity
able to be reduced and then oxidized
Binding modes of enzymatic catalysis
- Proximity effect
correct position decreases entropy, increase in reaction - Transition State Stabilization
Substrates bind weakly to enzymes
- not too tight, otherwise get a thermodynamic pit
- coenzymes are held a bit more tightly
- formation of ES is slightly unfavorable
Induced fit
- not rigid structures, flexible
- shift from inactive to active conformations
- activation by substrate induced change is called INDUCED FIT
- example: hexokinase
Transition State Stabilization
- process of distorting or straining a substrate towards the reaction’s transition state conformation
- lock and key mechanism
Transition State Analogs
- stable compounds whose structures resemble presumed transition states
- sit in active site of enzyme
- often potent inhibs
Trans State Analogs: Adenosine Deaminase Inhibs
enzyme recognizes the -OH at the C6 position binding to adenosine deaminase
Lysozyme
- Catalyzes the hydrolysis of polysaccharides that make up the cell wall.
- MurNAc -beta (1–4) -GlcNAc glycosidic bond
- substrate region can hold 6 saccharide units MurAc has to distort into a half chair to fit
Serine Proteases
- cleave the peptide bond of proteins
- essential serine residue in their active sites
- Tryspin for example cleaves at the COO- side of lys and arg residues
- there are three interesting ones–trypsin, chymotrypsin, elastase
Chymotrypsin
three catalytic residues– His 57, Asp 102, Ser 195
catalytic triad!!
Turns Ser 195 into a strong nucleophile
Enzymatic Cofactors
- assist in the catalytic process but aren’t enzymes
- Apoenzyme (inactive) and Holoenzyme (Apoenzyme and cofactor (active))
Essential Ions
- needed for metal activated enzymes
K, Ca, Mg - metalloenzymes
Zn, Fe, Cu, Co
Metalloenzymes
Zn can turn water into a good nucelophile
Iron-Sulfur clusters can accept a single electron
Coenzymes
Cosubstrates–substrates are chemically altered and dissociate with the enzyme
Prosthetic groups–remain bound to the enzyme throughout the reaction must be regenerated to retain enzymatic activity
Vitamin
- water soluble (B vitamins, vitamin C, folate)
- fat soluble (vitamin A, D, E, K)
excessive intake is toxic!
Adenosine Triphosphate
ATP is one of the most abundant coenzymes.
**Cosubstrate
*water
versatile reactant that can donate Pi, PPi AMP or adenosyl group
Nucleotide Cosubstrates
- S-adenosylmethionine is biological methylating agent
- Uridine diphosphate glucose (UDP) glycosyl donor
Nicotinamide coenzymes
NAD+ and NADP+ ****B3 **Cosubstrate **Active region is the nicotinamide *water can accept pair of electrons/ a hydride get reduced which makes them have an 'H' on the end
Flavin adenine dinucleotide and flavin mononucleotide
FAD and FMN ***both from riboflavin **Prosthetic group **semiquinones *water prosthetic group for many oxidoreductases -addition of proton and pair of electrons (usually hydride) -get or lose ONE ELECTRON at a time -semiquinones
Coenzyme A
involved in acyl group transfers
- **Vitamin B5
- *Active region is the free –SH group (CoA–SH)
- *Cosubstrate
- water
Thiamine Pyrophosphate
- **derived from thiamine (vitamin B1)
- *Active region is Thiazolium ring
- *Prosthetic
- water soluble
- coenzyme form is TPP
- several decarboxylase require TPP as a coenzyme (prosthetic group)
Pyridoxal Phosphate
- **Vitamin B6 is pyridoxal (or pyridoxamine)
- *pyridoxal 5’-phosphate (PLP) is active ALDEHYDE
- *Prosthetic
- water soluble
- PLP is a prosthetic group for enzymes that catalyze a variety of AA transformations including: isomerizations, decarby, eliminations, displacements
- works as a Schiff base
Transminase Mechanism
- PLP mechanism
- catalysis comes from transaminase but product comes from active site or near it
Tetrahydrofolate
- **folate, (vitamin B2 or folic acid)
- *two amine groups
- *Cosubstrate
- water
- needed by enzymes that perform one carbon transfers
Cobalamin
- **Cobalamin (vitamin B12)
- *Co atom
- *Prosthetic group
- water soluble
- corrin ring structure and a hexavalent Co atom ion
- coenzyme
Lipoamide
- **lipodic acid
- *Double SH groups
- *Prosthetic
- water
- swinging arm transfers acyl groups
- SH ester friends
Vitamin A
- Beta carotene
- **retinol
- *Prosthetic
- lipid
- retinal–prosthetic group for rhodopsin that makes neural impulses
- -retinoic acid which is a signaling molecule
Vitamin D
- calciferols
- **Vitamin D3 formed in the skin when exposed to sunlight
- lipid
Vitamin E
- helps to scavenge free radicals and oxygen
- *phenol group is active
- lipid vitamin
Vitamin K
- Helps in formation of proteins for blood coagulation
- *hydroquinone is active
- lipid vitamin
Ubiquinone
- coenzyme Q
- lipid membranes
- strong oxidizing agent can accept either one or two electrons
Protein Coenzymes
-Thioredoxins
two cysteine residues separated by two aminos
disulfide bridge exposed = reducing enzyme
-Cytochromes
Heme containing Fe atoms undergo one electron reduction
different types based on heme group structure
a,b,c based on absorbance
