10. Enzyme mechanisms Flashcards
what does an enzymes catalytic activity depend on
their native conformation
T or F: an enzymes catalytic activity can function when the enzyme unfolds
false; catalytic activity depends on their native conformation and will usually be lost if any degree of unfolding happens
where on the enzyme does the reaction take place
the active site
T or F: enzymes often require cofactors/coenzymes
true
what is a cofactor
a metal ion
what is a coenzyme
one or more organic/metalloorganic complexes, usually derived from vitamins
what do the coenzymes carry
a specific functional group
when the cofactor/coenzyme is tightly bound to the enzyme, what are they called
prosthetic group
what is free energy
the energy available to do work
describe the free energy in an exergonic reaction
free energy decreases
what does a reaction coordinate diagram look like
free energy vs progress of the reaction. There is a peak as S turns into P. There are troughs representing ES and EP
T or F: if a reaction is exergonic, it will be fast
false; just because something is spontaneous doesn’t mean it will be fast
what is a transition state
it’s the tall peak on a graph, and it represents an energy barrier as we go from S to P
what is the activation energy (Ea)
the difference between transition state and ground state of S
T or F; at the transition state, progress towards S or P is equally likely
true
explain why progress towards S or P is equally likely at the transition state
aligning groups and bond breakage have occurred to a point where decay to the products or decay to the substrates have the same odds
what does ∆Gǂ measure
∆Gǂ is the activation energy for the reaction. It measures the difference between the energy levels of the ground and transition states
what does a high activation energy mean
slower reaction
in regards to ∆Gǂ, what is the role of the enzyme
to decrease ∆Gǂ by lowering the transition state
why is a lock and key a bad analogy for substrates and enzymes
if the substrate perfectly fit into the enzyme would stabilize the substrate, and a product would not form. It would increase the activation energy needed to form P
what is binding energy (∆GB)
the free energy released from ES interactions in the transition state. It is the difference between the cat. and uncat. reactions
T or F: if there are multiple reaction intermediates, overcoming each barrier may require a dif. activation energy
true
which step is the rate limiting step
the step with the highest activation energy (has the highest peak)
what are the 6 categories of enzymes
oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases
describe oxidoreductases
catalyze redox reactions, most in this group are dehydrogenases, oxidases, peroxidases, oxygenases, or reductases
describe transferases
catalyze group transfer reactions (ie kinases)
describe hydrolases
catalyze hydrolysis, are special class of transferases with water serving as the acceptor of the group transferred
describe lyases
catalyze lysis of a substrate which generates a double bond or a ring
describe isomerases
catalyze structural changes within a single molecule to produce an isomer
describe ligases
catalyze ligation or joining of two substrates (usually linked to the breakage of a pyrophosphate bond)
what type of amino acids is the active site lined with
hydrophobic ones
why is the active site lined with hydrophobic amino acids
they help stabilize substrates through hydrophobic interactions
which residues in an enzyme are directly involved in the catalysis (there’s 5)
His, Asp, Arg, Glu, and Lys
what is the role of His, Asp, Arg, Glu, and Lys in catalysis
they function as acid, base, and nucleophiles, and they’re more likely to bind and stabilize transition states and cofactors
other than binding energy, how do enzymes promote catalysis
once an S is bound to E, properly positioned functional groups in E aid in the cleavage and formation of bonds to form P
name the 3 mechanisms for catalysis
- general acid base catalysis
- covalent catalysis
- metal ion catalysis
describe general acid-base catalysis
it’s a proton transfer mediated by weak acids and bases (not water). These acids/bases are amino acid residues within the active site
describe covalent catalysis
a transient covalent bond is formed between E and S. The side chain of the involved amino acid in the active site is either a nucleophile or an electrophile
describe metal ion catalysis
metals can either be tightly bound as cofactors or taken up from the solution along with the substrate. Ionic interactions between the metal and S can help orient S for the reaction
what are serine proteases
a class of enzymes that cleave peptide bonds. They’re characterized by the presence of catalytic serine residues within their active sites
give 3 examples of serine proteases
chymotrypsin, trypsin, and elastase
where are the serine proteases synthesized
in the pancreas
in what form are the serine proteases stored
stored as zymogens
list the names of the serine proteases when they’re in their zymogen form
chymotrypsinogen, trypsinogen, and proelastase
why are the serine proteases stored as zymogens
so they’re not degrading protein when they’re not supposed to
T or F: trypsin, chymotrypsin, and elastase are homologs
true
explain how the serine proteases are homologs
all 3 have a two-lobed structure with the active site in a cleft between the two domains. The positions of the catalytically active side chains (Ser, His, and Asp) are almost identical
describe how chymotrypsin’s active site differs from the others
it catalyzes cleavage of peptides on the carbonyl side of uncharged residues with aromatic or bulky hydrophobic side chains
describe how trypsin’s active site differs from the others
catalyzes cleavage of peptides on the carbonyl side of Arg and Lys
describe how elastase’s active site differs from the others
catalyzes the cleavage of elastin, a fibrous protein rich in Gly and Ala residues
describe the structure of chymotrypsin
3 polypeptide chains linked by disulfide bonds. Has a hydrophobic pocket where the bulky amino acid side chain of S will bind
what are the 2 reaction phases of chymotrypsin
acylation phase
deacylation phase
what is the acylation phase
peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme (specifically serine)
what is the deacylation phase
the ester linkage is hydrolyzed and a non acylated enzyme is regenerated
what are the 3 catalytic residues of chymotrypsin
His57, Asp102, Ser195
what do the 3 catalytic residues of chymotrypsin form
a hydrogen bonding network called the catalytic triad
describe the location of the H bonds within the catalytic triad
Asp with His, which is bonded to Ser
T or F: Gly is in the catalytic triad
false
since Gly isn’t in the catalytic triad, what is it’s role
it has a backbone N that contributes to an H bond to the tetrahedral intermediate
what nucleophile starts the chymotrypsin mechanism
the deprotonated oxygen of the serine OH group
where will the substrate be cleaved
on the carboxyl side of the residue in the middle
what is an oxyanion hole
an oxyanion is an oxygen anion (O-) so O- will go in the hole
describe what happens when a substrate binds to chymotrypsin
causes a conf. change, bringing Asp and His closer together, compressing the H bond between them. Now, His REALLY wants a proton, and it will get it from the OH group of Ser. Also, the side chain of the residue adjacent to the pep bond nestled in a hydrophobic pocket
what effect does deprotonation have on Ser
it makes it a really strong nucleophile
see notes for the rest of chymotrypsin mechanism
why do enzymes have a pH range where their activity is maximal
as pH varies, dif. R groups may change their ionization status
what pH is chymotrypsin’s peak velocity around
8
T or F: both kcat and Km will be affected by pH changes
true
what affect will a protonated His have on kcat
it will lower the kcat because when it’s protonated it no longer needs to steal protons from Ser
Km is ___ at low pH values
low
what changes as Km increases as pH increases
polypeptide B of chymotrypsin has Ile16 at the N term, and it normally forms a salt bridge with Asp194 when charged. The salt bridge stabilized the active site, so when you protonate you lose the bridge and therefore stabilization (this collapses the hydrophobic pocket)
