biochem 13-15 protein dynamics

what is a catalyst

a compound that increases the rate of a chemical reaction

catalyst to what to activation free energy (ΔG)

lowers

what is activation energy

the difference in energy between the reactant in its ground state and in its transition state

what is transition state

"setting up the reaction" by breaking existing bonds and forming new ones

catalysts combine transiently with what, which promotes what

• They combine transiently with the reactants promoting a reactive transition state condition

what does catalyst do to ΔG

does not alter

average energy of reaction does not change

enzymatic catalysis offers what 3 things

• Acceleration under mild conditions

• High specificity

• Possibility for regulation

no enzyme vs enzyme catalyzed reaction graph

enzymes catalyze what types of reactions; causes them to do what

Thermodynamically favorable reactions

• Causing them to proceed at extraordinarily rapid rates

• Living systems use enzymes to accelerate and control the rates of vitally important biochemical reactions

enzymes and metabolic function

Enzymes are the agents of metabolic function

the reactants in an enzyme catalyzed reactions are referred to as

substrates

what is catalytic power

• Catalytic power is defined as the ratio of the enzyme-catalyzed rate of a reaction to the uncatalyzed rate

  • Relationship between catalyzed reaction and the uncatalyzed, how much faster is reaction with enzyme compared to without enzyme

• Enzymes can accelerate reactions as much as 10^26 over uncatalyzed rates

• Urease is a goof example

  • Catalyzed rate: 3x10^4/sec

  • Uncatalyzed rate: 3x10^-10/sec

  • Ratio (catalytic power) is 1x10^14

specificity

is the term used to define the selectivity of enzymes for their substrates

regulation of enzyme activity ensures

• that the rate of metabolic reactions is appropriate to cellular requirements 

  • Not break down all we are making before we get to use it

what are coenzymes and cofactors

• are nonprotein components essential of enzyme activity

  • Vits, minerals, ATP, NADH

  • Not in the amino acid sequence but needed for the protein function (enzyme)

enzyme specificity

• Enzymes selectively recognize proper substrates over other molecules

  • Produce products in very high yields- often much greater than 95%

specificity is controlled by

structure

The unique fit of substrate with enzyme controls (2)

The unique fit of substrate with enzyme controls the selectively for substrate and the product that is formed

what are the major classes of enzymes (6)

• Oxidoreductases - oxidation reduction reactions

• Transferases - transfers a functional group between 2 compounds

• Hydroxylases- hydrolyzes (adds water to split molecule)

• Lyases- break it by removing something

• Isomerases - making isomer

• Ligases  - combining things using ATP

what is a cofactor

Inorganic ions (Fe+2, Mg+2, Zn+2)

what is a coenzyme

• A complex organic or metalorganic molecule that act as a carrier of functional groups (biotin in carboxylation reaction)

• Usually derived from vitamins

coenzymes and cofactors are __________ changed

reversibly

• They need to be able to go back to original state to be able to redo reaction

• So like F2+ became F3+ after reaction, so then needed to go back to F2+ to be reused

what is a prosthetic group

A coenzyme or metal ion tightly or covalently bound to the enzyme (heme)

what is a haloenzyme

•  enzyme together with its bound coenzyme/metal ion (glutathione reductase with Se-)

  •  enzyme is whole so it has its cofactor or coenzyme

what is apoenzyme/apoprotein

• the protein part of an enzyme (lipoproteins)

  • without its cofactor or coenzyme

covalent modification

• Covalent attachment via phosphorylation, glycosylation, etc.

•  alters enzyme activity or provides regulation

now onto kinetics …

what is kinetics (and what is its 2 goals)

 is the study of the rate at which compounds react

• Enzymes kinetics seeks to determine the max reaction velocity that enzymes can attain

• and the binding affinities for substrates and inhibitors

what effects the enzymatic reaction rate (3)

• Enzyme

• Substrate

• Temperature - slow down at cold and speed up at warm, yet too high denatures

why do we measure enzymatic kinetics (5)

• Quantitative description of enzymes

• Determine the order of binding of substrates (like in metabolic pathways )

• Understand catalytic mechanism

• Find effective inhibitors (or activators)

• Understand regulation of activity

 

what is E, S, P, K

enzyme

substrate

product

rate of reaction

expression (equation of enzymes)

equation if done at initial rate

(before high [P])

• Look at the initial rate because at the beginning you have more energy, if we do it later than most energy already spent (like class at 9:30am vs class at 2:30pm)

how do we do analysis of initial rate

• Mix the same [E] but varying [S]

• Measure initial rates of [S] disappearance

• Plot change in [S] vs time

• Calculate initial rate based on slopes

For E +S <--> ES→ E+P concentrations vary of time for (which parts of the equation)

• Substrate [S]

• Free energy [E]

• Enzyme-substrate [ES]

• Product [P]

steady state in enzyme kinetics

• After a very brief initial period, [ES] reaches a steady rate

  • ES is consumed approximately as fast as it is formed

  • Allows us to calculate the velocity by assuming steady-state conditions

• WE STUDY THE INITIAL RATE BECAUSE OTHERWISE THE ES IS STEADY, AND WE CANT REALLY OBSERVE ANYMORE WHAT IS HAPPENING

michealis menton equation has what parts

Km and Vmax

what is Km

• Michaelis constant

  • Specific for each E acting on a given S

  • Equal to substrate concentration is 1/2 the Vmax

Vmax

• maximum velocity

  • Point at which E is saturated

Km is the [S] when V is what

1/2Vmax

when [S] is above Km, enzyme activity is

high

Km is also knows as

kinetic activator constant

small Km means ______ affinity

• Small Km means high affinity of the enzyme for its substrate

  • Very sensitive to low amounts of that substrate

  • Needs less substrate to reach Vmax than if Km is high

high Km means ____ affinity

• High Km means low affinity  of the enzyme for its substrate

  • Needs a higher amount of substrate present to reach Vmax

can Vmax be reached

• Vmax is the theoretical maximal rate of the reaction- but it is NEVER achieved in reality

  • To reach Vmax would require that ALL enzyme molecules have substrate bound

  • Vmax is asymptotically approached as substrate is increased

how does pH affect enzyme activity

• Enzyme- substrate recognition and catalysis are greatly dependent on pH

• Enzymes are usually active only over a limited range of pH

• The effects of pH may be due to effects on Km or Vmax or both

how does pH effect structure and active site

Enzymes have a variety of ionizable side chains that determine their secondary and tertiary structure and also affect events in the active site—> so pH can mess with the side chains and therefore mess with structure and the active site

how are substrates affected by pH

they also may have ionizable groups

• and remember pH messes with this (tritation of amino acids)

how does temp affect enzyme

• Rates of enzyme-catalyzed reactions generally increase with increasing temperature

• However, at temperatures above 50° to 60°C, enzymes typically show a decline in activity --> because of denaturing

what 2 effects does temp have

• Enzyme rate typically doubles in rate for every 10ºC rise in temperature as long as the enzyme is stable and active (as long as hasn’t denatured)

• At higher temperatures, the protein becomes unstable and denaturation occurs

what are the 2 types of inhibitor classes

• inhibitors (noncovalently bound)

  • Can be removed or come and then leave

• Irreversible inhibitors (covalently bound)

  • Cannot be removed  

what are the 3 types of reversible inhibition

competetive, uncompetivite, mixed

competitive inhibitors

• Competes with substrate for the active site

  • Binds to active site (competes for the active site)

  • Does not effect catalysis

• (does not change the Vmax, but increases km)

• What happens is that in order to get to same rate as with no inhibitor, you will need to add a lot more substrate

uncompetitive inhibition

• Only binds to ES complex

  • Does not affect substrate binding(binds somewhere else on the enzyme)

  • Only binds once the substrate binds to the enzyme

  • Inhibits catalytic function

    • Decreases Vmax  (overall catalytic activity) but Km of enzyme stays the same

    • noncompetitive inhibition reduces the overall catalytic activity of the enzyme by affecting its function, but it does not prevent substrate binding. This leads to a lower Vmax while leaving the Km unaffected.

mixed inhibition

• Binds to enzyme with or without substrate

  • Binds to regulatory site (not the active site)

  • Inhibits both regulatory binding and catalysis

    • Decreases Vmax and Km

      • If binds before substrate binds to enzyme, then changes active site

      • If binds to the ES at secondary site, then affects catalysis because not binding active site

irreversible inhibition

• Substances combine covalently with enzymes→ toxic

  • irreversible inhibition

  • React with functional group in active site

  • Many are transition state analogs

what are the enzyme substrate models

lock and key

induced fit

lock and key hypothesis

the first explanation for specificity (explains substrate specificity)

does not explain catalysis or transition state

induced fit hypothesis

• provides a more accurate description of specificity

  • Induced fit favors formation of the transition state

  • allows catalysis to occur (speed up reaction and lower activation energy)

hexokinase and specificity

• Specificity and reactivity are often linked

  • In the hexokinase reaction, binding of glucose in the active site induces a conformational change in the enzyme that causes the 2 domains of hexokinase to close around the substrate, creating the catalytic site

are all enzymes proteins

no

robozymes

•  RNA molecules that display enzyme activity in the absence of protein

  • Examples: RNase P and peptidyl transferase

abzymes

antibodies raised to bind the transition state of a reaction of interest

The large rate accelerations of enzymes correspond to (what does enzyme do that increases rate fo reaction)

large decreases in the free energy of activation for the reaction

all reactions pass through a _____________________________ on the reaction pathways

transition state

what is the transition state

state of most free energy required

not the product yet, but changes occurring to drive to the product

active sites of enzymes bind more tightly to what than what; why?

• The active sites of enzymes bind the transition state of the reaction more tightly than the substrate

  • It prefers the transition state over the substrate because this will help drive the reaction, if it binds more to the substrate then no reaction would occur.

the active sites of enzymes binding more tightly to the transition state than the substrate does what

the enzymes stabilize the transition state and lower the activation energy of the reaction

The catalytic role of an enzyme is

 to reduce the energy barrier between substrate S and transition state X‡

what does X‡ represent

transition state

what does rate acceleration of an enzyme mean

rate acceleration by an enzyme means that the energy barrier between ES and EX‡ must be smaller than the barrier between S and X‡

• This means that the enzyme must stabilize the EX‡ transition state more than it stabilizes ES

If we were stabilizing ES, then nothing would happen, by moving it to transition state, this helps drive it forward by starting to change it which them moves it to continue changing until reaching the EP

how does formation of ES help with catalysis (in terms of transition state)

For a given energy of X‡, raising the energy of ES will increase the catalyzed rate

raising the energy of ES will _____the catalyzed rate

increase

HOW TIGHTLY DO TRANSITION-STATE ANALOGS BIND TO THE ACTIVE SITE?

• Very tight binding to the active site

• The binding constant of the enzyme for the transition state may be 10^ -20 to 10^-26 M!

Transition state analogs (TSAs)

stable molecules that are chemically and structurally similar to the transition state

• That mimic the transition state

• We use these because the enzyme prefers the transition state over the substrate—> enzymes will allow them to bind over substrate because they are already stable

• Would be very good competitive inhibitors

• Proline racemase was the first case observed

Transition-state analogs work well as what

enzyme inhibitors

-competitive

-irreversible

what 5 things that will cause enzymatic catalysis (one or more of these is needed) (5)

• General acid/base catalysis (GABC)

• Covalent catalysis

• Electrostatic stabilization (Positive charge on enzyme helps stabilize the neg charge on substrate)

• Proximity effects

• Preferential stabilization of the transition state

what else may play a role in catalysis

protein conformational changes

what is essential to enzyme catalysis

protein motions

protein movement (and importance for catalytic events)

• Proteins are constantly moving –

  • Bonds vibrate, side chains bend and rotate, backbone loops wiggle and sway, and whole domains move as a unit

  • Enzymes depend on such motions to provoke and direct catalytic events

Active site conformation changes can - 4

Active site conformation changes can

• Assist substrate binding

• Bring catalytic groups into position

• Assist in bond making and bond breaking

• Facilitate conversion of substrate to product

HOW DO ACTIVE SITE RESIDUES INTERACT TO SUPPORT CATALYSIS

(main effect, secondary roles (4)

• About half of the amino acids engage directly in catalytic effects in enzyme active sites

• Other residues may function in secondary roles in the active site:

  • Raising or lowering catalytic residue pKa values

  • Orientation of catalytic residues

  • Charge stabilization

  • Proton transfers via hydrogen tunneling

active site has what 2 parts

binding sites and catalytic sites

bind vs catalytic site

• Blue is secondary roles

• Red is actually catalytic activity place

what factors influence enzymatic activity (4)

The availability of substrates and cofactors

Genetic regulation of enzyme synthesis and decay

allosteric or covalent modification

Zymogens, isozymes, and modulator proteins

how does availability of substrates and cofactors effect enzyme activity

• usually determines how fast the reaction goes

  •  As product accumulates, the apparent rate of the enzymatic reaction will decrease

how does Genetic regulation of enzyme synthesis and decay effect enzymatic activity

determines the amount of enzyme present at any moment

what are the 2 ways enzyme activity can be regulated

allosterically or covalent modification

zymogen

• inactive precursors of enzymes. Typically, proteolytic cleavage produces the active enzyme

  • So pretty much inactive enzyme that is activated by some sort of modification

REGULATORY CASCADE OF BLOOD CLOTTING

• Soluble fibrinogen is a zymogen that must be cleaved to form insoluble fibrin by thrombin

• Some factors and proteins have Ca2+ binding sites which were formed via PTM of a Glu residue by Vitamin K dependent gamma -glutamy carboxylase

  MAIN THING: We need PTMs and zymogens for our blood to clot - main point

ISOENZYMES

ENZYMES WITH SLIGHTLY DIFFERENT SUBUNITS

• Play same role but differ slightly depending on where they're made in the body

• they often respond to different inhibitors and activators (was on slido)

  • Because of changes in subunits they may respond to different things

what is allosteric regulation

• Action at "another site"

does not bind to active site to regulate

enzymes in metabolic pathways are modulated by

allosteric effectors

the effectors usually made where, and what roles do they play (2)

• These effectors are usually produced elsewhere in the pathway

• Effectors may be feed-forward activators or feedback inhibitors

the effectors are usually products in some part of the pathway or result of lack of product

what is the most common covalent modification used to regulate activty of enzyme

• reversible phosphorylation

  • This is the MOST prominent form of covalent modification in cellular regulation

phosphorylation (in terms of regualtion)

• Phosphorylation is accomplished by protein kinases

  • Each protein kinase targets specific proteins for phosphorylation

  • Phosphoprotein phosphatases catalyze the reverse reaction – removing phosphoryl groups from proteins

protein kinases

 targets specific proteins for phosphorylation

Phosphoprotein phosphatases

catalyze the reverse reaction – removing phosphoryl groups from proteins

Protein kinases phosphorylate what residues in target protein

• Ser, Thr, and Tyr

kinases recognize what in their target

amino acid sequence

• Despite this specificity, all kinases share a common catalytic mechanism based on a conserved core kinase domain of about 260 residues