Lecture 8: Enzyme Kinetics (not reviewed)

True or false: the enzyme is a better fit for the transition state than for the substrate itself and thus lowers the activation energy

True

What is differential binding

enzymes binding to substrates through induced fit and thus contorting the substrate into a structure more similar to the transition state

What is binding energy and why is it important

• Binding energy is the free energy released when an enzyme binds its substrate weak interactions

• then stabilizes the transition state and lowers activation energy, speeding the reaction.

How does bidding energy influence enzyme specificity

it disallows the incorrect substrate from binding to an incorrect enzyme because the contacts necessary to achieve the necessary binding energy are to lower activation energy and stabilize the transition state

True or false: when a substrate is completely held in the active site, it take the characteristics of the transition state

True

True or false: Like ΔG, the rate of a reaction is constant

False, a rate is not constant, it changes over time

What are the phases in enzyme catalysis

1. Pre Steady State

2. Transition Point

3. Steady State

What is the pre-steady state

• initial transient period during which the concentration of the enzyme-substrate complex builds up as they form

• the enzyme is picking up substrates

What is steady state

• the period in which the enzyme-substrates complex an other intermediates remain constant

• the enzyme cannot pick up any more substrate

• concentration of ES is constant

• V₀ (initial velocity)

What is the timescale of the pre-steady state

microseconds

What is the timescale of the steady state

seconds to minutes

Where does Micheal’s Menten live?

at the steady state!!!

What is initial velocity (V₀)

the rate of the product formation when time is 0, and its so early before product accumulates

True or False: at the steady state the product can turn around and inhibit the formation of the ES complex

True

What happens other than velocity a we keep increasing the concentration of substrate

more ES complex is formed

What is Vmax

the maximum velocity the enzyme can achieve, where eery enzyme molecule is occupied

• not a true max, due to eq it is not possible to MAX

• initially btw

If the rate of increase of V₀ (initial velocity) slows what does that mean?

free enzyme is becoming scarce

What is a first order reaction

Rate = k[s], where th rate is linear with concentration of substrate (inc substrate inc rate)

What is a zero order reaction

Rate = k, where the rate is independent of the concentration of substrate

• no matter how much S you add, the rate is unfuckwitable

What is k1

the rate of the enzyme and substrate creating the enzyme-substrate complex, ES.

• binding!!

what is k_-1

the rate of the enzyme-substrate complex falling apart and disassociating into the E and S respectively

What is k2

the rate of the enzyme-substrate complex being catalyzed into the product.

What is Km

• the michealis menten constant, that is unique to each enzyme and is independnet of enzyme concentration

• tells us the affinity of an enzyme for its substrate

• substrate concentration at Vmax/2

What is the michealist menten equation and what does it describe

It is the Velocity at time zero = Vmax (conc of S) over Km + conc of S

What is the Lineweaver Burk-Plot

it is the Michealis-Menten Equation plot but linearized by taking the reciprocal of both sides

What are assumptions being made for the michealist menten equation

1. the reverse reaction (P —> E + S) doesn’t matter since the initial rate velocity is what we care about and at that point there are no products

2. The ES complex is a steady state intermediate so the concentration of ES stays relatively stable

Remember, with nichealis mentin, you’re looking at V0 which is th initial velocity where time is ZERO

ok past Asare

What is the slope of the Lineweaver-Burk Plot?

Km/ Vmax

What does the Y-intececpt of the Lineweave-Burk Plot tell you?

1/V_max

What does the X intercept of the Lineweaver-Burk Plot tell you?

the -1/K_m

what is turnover #

the number of S converted to P by E when fully saturated

What is an enzyme reaction involving a ternary complex ? (sequential)

A SEQUENTIAL kind of enzymatic reaction where there are multiple reactants and all substrates bind to their enzyme before any product is released.

Also, a ternary complex of the enzyme and both substrates form

What are the two kinds of enzymatic reactions involving a ternary complex?

1. ordered ( E+S eq ES₁ eq E₁S₂ —→E + P₁ + P₂

2. non-ordred same as above but circular

What happens to the lineweaver-burk plot when the enzymatic reaction involves ternary complexes

there will be INTERSECTING lines telling us that the reaction is sequential

What is the enzymatic reaction in which NO ternary complex is formed?

• Ping Pong/Double Displacement mechanism, a reaction having multiple substrates in which one or more products are released before all substrates bind the enzyme

• crank em out as quick as possible

What is a ternary complex

a complex with three parts, enzyme, substrate, and substrate

• seen in sequential enzymatic reactions

What happens to the Lineweaver-Burk plot one the enzyme and substate participate in ping pong/double displacement mechanisms

the lines become parallel

True or False: Michelias Menten enzymes are not passive and can be fine tuned signaling molecules

False, micheal’s menten genetic are passive and rely solely on concentration and in turn cannot be fine tuned via signaling molecules

True or false: Michealis Menten genetics do not apply to allosteric enzymes

True, allosteric enzymes are under different kinetic rules

True or False: Michealis Menten Genetics are ubiquitous, applying to all enzymes

FALSE

What differentiates allosteric enzymes from Michealis Menten enzymes

allosteric enzyme have as secondary regulator separate from the active site that allow the cell to turn enzyme activity up or down while MM enzymes cannot be regulated outside of concentration changes

What is an allosteric enzyme?

• a enzyme with an active site and allosteric site(s) that when bound changes the confirmation of the enzyme making it high or low affinity thus regulating enzymatic activity

What are homotropic enzymes

homotropic enzymes are allosteric enzymes where both th active site and allosteric site bind to the same substrate

What are heterotropic enzymes?

heterotropic enzymes are allosteric enzymes where the active site and allosteric site bind to different substrates

What is feedback inhibition

a mechanism in allosteric enzyme pathways where the product(s) of the pathway inhibits one enzymatic reaction and thereby regulates the pathway

What are the two kinds of allosteric enzyme regulation

1. reversible and non reversible

2. where non covalent binding is reversible but covalent modifications can be irreversible OR reversible

Allosteric enzymatic pathways depend on alteration in the quaternary strcutre

ok!

What are the two models for cooperative binding

1. Concerted Model

2. Sequential Model

What is cooperative bidding

• something that occurs only in allosteric enzymes

• when something bids to the allosteric site taking the confirmation from Tense ( T, not enzymatically active) to Relaxed (R, enzymatically active)

What is the concerted model for cooperative binding

the model for cooperative ending that says that all active site must be in the same state at the same time

all R or all T no hybrid

What are the R and T confirmations and what do they mean?

R is relaxed and allows for pro eenzymatic activity, while T is tense and disallows enzymatic activity

What is the sequential model for cooperative binding

the model for cooperative ending intact says that allotter centimes can undergo sequential changes in structure so you can have hybrids of R and T

You see a signmoid curve when you have what type of enzyme?

allosteric

What are the chacateristcis of a allosteric enzyme graph plot?

one curve showing low activity (tense), one showing high activity (R)

What are allosteric regulators

small molecules that can be positive or negative thus shift the sigmoidal curve