lecture 3: enzymes Flashcards
what are enzymes
- Enzymes are proteins or RNA molecules (ribozymes) that are powerful and specific
what do enzymes change and keep the same in any reaction
They speed up the forward and reverse reactions and do not change the Keq, the free energy change of the reaction, ΔG, nor the standard free energy change, ΔGo.
Mechanism of Enzyme Catalysis: binding
Enzymes contain an active site, which binds substrate, the transition state, and product with multiple noncovalent interactions. As the substrate binds, the enzyme conforms around it (induced fit).
how does the active site of the enzyme make the change to the product more likely?
The active site reduces the free energy difference between the transition state and the substrate ( Ea).
what are the ways that the enzyme, at the active site, reduces the Ea (physically speaking)?
a. Gathering substrates together in favorable orientation (proximity and orientation)
b. Creating a microenvironment to favor catalysis (e.g. nonpolar, acid-base, metal ion cofactor, or temporary covalent attachment)
c. Positioning of R groups such that the transition state has the greatest binding energy and is, therefore, stabilized
cofactors
Some enzymes require cofactors (metal ions or coenzymes such as NAD+) to accomplish catalysis.
holoenzyme
enzyme + cofactor
prosthetic groups
Coenzymes that are always bound to the active site are called prosthetic groups.
Reaction Velocity
product formed (or substrate lost) per time (e.g., micromoles of carbonic acid formed per minute by the action of carbonic anhydrase
Zero order reaction velocity: substrate
velocity doesn’t change as [substrate] changes
First order
a. velocity changes linearly with [S]
Second order
velocity changes with the product of [A] and [B] or [A] x [A]
enzyme kinetic plot- what order is it?
starts out first order and ends up zero order as [S] increases.
Michaelis and Menton worked out a different formula based on the idea that an enzyme-substrate complex forms (ES) and thus:
the concentration of enzyme available limits the velocity when all sites are temporarily occupied by substrate (maximum velocity, Vmax).
definition of Km
a measure of the affinity of the enzyme for its substrate. High Km= low affinity
Vmax depends solely on the ___
enzyme
KM is the substrate concentration at which Vo =
½ Vmax
The maximum velocity, Vmax, is achieved when all enzymes are in ___
the enzyme-substrate form
turnover number
how fast one enzyme converts substrate to product, e.g. carbonic anhydrase is 600,000 molecules per second
The enzyme efficiency
kcat/Km, taking into account both speed and affinity. This parameter is used to compare which substrate the enzyme prefers or which isozyme is the most efficient at catalysis, for example
Lineweaver-Burke
plot of 1/Vo and 1/[S], which yields a straight line
Competitive Inhibition
the inhibitor binds to the active site and blocks the substrate
what changes/stays same in competitive inhibition
Km is higher and Vmax is unchanged because high [S] swamps out inhibitor effect.
Noncompetitive Inhibition
the inhibitor binds to either E or ES and blocks catalysis. ‘changes shape’
what changes/stays same in allosteric inhibition?
a. Vmax is lower and Km is unchanged because active [enzyme] is lower due to [ESI] or [EI].
Uncompetitive Inhibition
the inhibitor binds to ES and blocks catalysis
what changes/stays same in uncompetitive inhibition
Km is lower because of LeChatlier’s principle: formation of ESI depletes ES and draws E + S to form more ES, which appears the same as higher affinity of E for S (i.e., lower Km). The percent of E removed to ESI appears that the [enzyme] is lower so Vmax is also lower.
describe an allosteric enzyme, what it looks like and its conformations
All have multiple subunits (tetramers) with active sites and regulatory sites. The subunits have at least two conformations termed T for tense and R for relaxed
T vs R states and what they mean
The T state has less activity and less affinity for Substrate than the R state.
T/F: allosteric enzymes do not conform to michaelis-menten kinetics
True; display an s –shaped curve
cooperative’ behavior
noncompetitive behavior
why does the s-shaped curve look that way?
At low substrate levels, has a hard time getting started. As s increases to an intermediate level, zoomed up- velocity increases GREATLY in response to a tiny change
S-shape of curve is due to ___ effect
homotropic
homotropic effect vs heterotropic
Homotropic is where the molecule that induces the effect is similar to the substrate
Heterotropic is when the molecule that induces the effect is different from substrate.
What are the two models of homotropic effectors?
1) Concerted - dimeric enzyme, binding of first substrate has effect on binding of second substrate
2) Sequential -four subunits, binding of 1st substrate facilitates binding of following substrates
Inhibitors: stabilize __ states
Activators: stabilize __ state
T, R
how are allosteric enzymes regulated?
by feedback inhibition; regulated by one or more end products of the pathway or pathways under their control
allosteric enzymes catalyze the ___ step in a pathway
committed
Positive feedback-
Two pathways cooperate to form a single product
homeostasis
Levels of intermediates in these pathways always stay around the same
T/F: T and R are in equilibrium
True
T/F: R state is more enzymatically active than T
true
concerted model
If a substrate binds to the 1 R one, whole thing turns into R. (all 4 units) now 4 R’s .
Sequential model:
when one substrate binds, all the adjacent subunits flip shape too. But it doesn’t require that they shift AT THE SAME TIME (in concert); they can shift in a sequential manner. One by one
the disruption of the T/R equilibrium by substrates
homotropic effect
disruption of T/R equilibrium by regulators
heterotropic effect
effect of regulators on the s curve?
shift it one way or the other - activator increases it, T state/inhibitor decreases it
do all enzymes take on the same conformations?
no; MM curves are averages of all enzyme activity
a___inhibitorcan bind whether or not the substrate is bound,
noncompetitive
Uncompetitive: ONLY BINDS TO :
E-S complex
When delta G is positive, the reaction does not occur spontaneously, and is:
endergonic
standard Gibbs free energy of formation
The standard Gibbs free energy of formation of a compound is the change of Gibbs free energy that accompanies the formation of 1 mole of that substance
