mid. 2

Enzymes are the agents of _____
metabolic function
Enzyme: a biological _____
catalyst
Enzyme: a biological catalyst
with the exception of some____ that catalyze their own splicing (Section 10.4), all enzymes are_____
RNAs. proteins
enzymes can ____ the rate of a reaction by a factor of up to _____ over an uncatalyzed reaction
increase. 10 to the 20th
enzymes can ____  the rate of a reaction by a factor of up to ____ over an uncatalyzed reaction
increase. 1020

some enzymes are so ____  that they catalyze the reaction of only one ____; others catalyze a family of similar reactions

specific. stereoisomer
Enzymes produce products in very _____ -> often much greater than ____
high yields. 95%

Enzymes can accelerate reactions as much as 1020 over uncatalyzed rates!

 

Urease is a good example:

Catalyzed rate: 3×104/sec

Uncatalyzed rate: 3×10 -10/sec

Ratio is:  !

1 X 1014
 

D-amino acid oxidase only oxidizes ____ and not ____ .

D-amino acids.  L-amino acids
[image]
look at me again
[image]
look at me again
 

The breakdown of glucose by ___  provides a prime example of a metabolic pathway. Ten enzymes mediate the reactions of glycolysis.

glycolysis
 

Enzymes are highly regulated at

      the ___ level

      the ____ level

      the ____  level

activity

protein

gene

 

Oxidoreductases

Act on many chemical groupings to add or remove ____  atoms or ______.

hydrogen

electrons

Oxidoreductases
Spell it
 

Transferases

Transfer functional groups between ___  and _____  molecules.

donor

acceptor

Kinases are specialized ____  that regulate metabolism by transferring ______  from ATP to other molecules.

transferases

phosphate

 

Transferases

Spell it
 

Hydrolases

Add water across a _____ , hydrolyzing it.

bond
Hydrolases
Spell it
 

Lyases

Add ____,  _____ or _____ across double bonds, or remove these elements to produce double bonds or other cleavages involving electron rearragenement.

 

water

ammonia

carbon dioxide

 

Lyases

 

spell it
 

Lyases

Add water, ammonia or carbon dioxide across ____ , or remove these elements to produce ______or other cleavages involving _____ rearragenement.

 

double bonds

double bonds

electron

 

Isomerases

Carry out many kinds of isomerization: ____ isomerizations, mutase reactions (shifts of chemical groups) and others.

L to D

 

Isomerases

Spell it
 

Isomerases

Carry out many kinds of isomerization: L to D isomerizations, ___ reactions (shifts of ____ ) and others.

mutase

chemical groups

 

Ligases

Catalyze reactions in which two chemical groups are  ____ (or ligated) usually with the use of energy from ____.

joined

ATP

 

Act on many chemical groupings to add or remove hydrogen atoms or electrons.

Oxidoreductases

 

Transfer functional groups between donor and acceptor molecules.

Transferases

Add water across a bond, hydrolyzing it.

Hydrolases

 

Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds or other cleavages involving electron rearragenement.

Lyases

 

Carry out many kinds of isomerization: L to D isomerizations, mutase reactions (shifts of chemical groups) and others.

Isomerases

 

Catalyze reactions in which two chemical groups are joined (or ligated) usually with the use of energy from ATP.

Ligases
 

Examples                  RXN

Oxidoreductasesà Alcohol Dehydrogenaseà turns ___ into ______.

Ethanol

Acetaldehyde

 

Transferasesà Hexokinase à turns _____ into _____

 

D-Glucose

D-Glucose-6-phosphate

Hydrolase à Carboxypeptidase à turns a molecule with a  _____ bond into a ____

 

double

single

Lyases à Pyruvate decarboxylase à turns ___  into ____

 

 

Pyruvate

Acetaldehyde

Isomerases à Maleate isomerase à Turns ___ into  ____

 

 

Maleate

Fumarate

Ligases à Pyruvate Carboxylase à Turns  ___ into ____.

Pyruvate

Oxaloacetate

  

_______à Alcohol Dehydrogenaseà turns Ethanol into Acetaldehyde

 

Oxidoreductases

_______à Hexokinase à turns D-Glucose into D-Glucose-6-phosphate

 

Transferases

 ______à Carboxypeptidase à turns a molecule with a double bond into a single

 

Hydrolase

 ______à Pyruvate decarboxylase à turns Pyruvate into Acetaldehyde

 

Lyases

 ______à Maleate isomerase à Turns Maleate into Fumarate

 

Isomerases

 _______à Pyruvate Carboxylase à Turns Pyruvate into Oxaloacetate

Ligases
 

ENZ.                 Optimum pH

 

Pepsin              _____

 

1.5

                     pH 

Catalase      _____ 

 

7.6

                 pH 

Trypsin     ______ 

 

7.7

                           pH 

Fumarase         ____

 

7.8

 

                                       pH

Ribonuclease               ____

Arginase                       ____

7.8

9.7

 

______ changes the rate of the catalyze reaction ( rate ___ as temperature goes up)

 

 

Temperature

increase

However, there is an optimum temperature, why?

 

• Increasing temperature will eventually lead to _______.

protein denaturation

 

Enzyme Kinetics

 Free Energy of RXN

For a reaction taking place at constant temperature and pressure, e.g., in the body→  _____.

 

                                 

 A <=> B

the change in free energy is represented by this rxn

___________________

ΔG°= ΔH° – TΔS°
 

 

The change in free energy is related to the equilibrium constant, Keq, for the reaction by

______________________________

 

ΔG° = -RT in Keq
 

An enzyme alters the rate of a reaction, but not its _____ change or position of _______

free energy

equilibrium

[image]

look at me again

 

The rate of a reaction depends on its______ , D

activation energy

 

The rate of a reaction depends on its activation energy, denoted as _____(or symbol is)?

D
 

a enzyme provides an alternative pathway with a ____ activation energy

lower
  left off on slide 16 on 06A enzymes I v 4.1 file

Understand the difference between ΔG0 and ΔG0

 

 The overall free energy change, ΔG0, for a reaction is related

to the ________.

equilibrium constant
The free energy of activation, ΔG0‡,  for a reaction is related to the _____.
rate constant
 

The overall free energy change, _____  for a reaction is related

 

to the equilibrium constant

ΔG0
 

The free energy of activation, ____ for a reaction is related

 

to the rate constant

 ΔG0‡, 
[image]
look at changes
 

         In an enzyme-catalyzed reaction  S → P

substrate, S: a ______

 active site: the small portion of the _______ where the substrate(s) becomes bound by ______ forces, e.g.,  ______bonding, _______attractions, van der Waals attractions

 

reactant

enzyme surface

noncovalent

hydrogen

electrostatic

 enzyme-catalyzed reaction  S → P

Specificity is controlled by _____ – the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield – all refered to as the  ______.

structure

ACTIVE SITE

[image]
 Two models have been developed to describe formation of the enzyme-substrate complex

 

lock-and-key model: substrate binds to that portion of the enzyme with a __________.

induced fit model: binding of the substrate induces a______ in the ______  of the enzyme that results in a complementary fit

complementary shape

change

conformation

 

Two models have been developed to describe formation of the enzyme-substrate complex

_____: substrate binds to that portion of the enzyme with a complementary shape

______: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit

lock-and-key model

induced fit model

[image]
[image]
Formation of a prodcut

 For the reaction–>

A + B —> P

The rate of reaction is given by rate equation—>

Rate = k[A] f[B]g

Rate = k[A] f[B]g

 

Where k is a proportionality constant called the specific ________.

Order of reaction: the ______ of the exponents in the rate equation .

rate constant

sum

File, Enzymes I 4.1. slide 24 ??
[image]
 

Chymotrypsin catalyzes the selective hydrolysis of peptide bonds where the carboxyl is contributed by Phe and Tyr

it also catalyzes hydrolysis of the ester bond of pnitrophenyl esters

[image]
 Non-Allosteric Enzyme Behavior

Chymotrypsin

 

Point at which the rate of reaction does ______, enzyme is _____ , maximum rate of reaction is _____.

 

not change

saturated

reached

[image]
 

Initial rate of an enzyme-catalyzed reaction versus substrate concentration…look at pic–>

 

[image]
Michaelis-Menten Model

 

for an enzyme-catalyzed reaction–> (rxn)–>

 

[image]
 

Michaelis-Menten Model

the rates of formation and breakdown of ES are given by these equations —->

[image]
 Michaelis-Menten Model

At the steady state the equation is–>

[image]

 

Michaelis-Menten Model

when the steady state is reached, the concentration of free enzyme is the total less that bound in ES—-represented by equation–>

 

[E]=[E]t-[ES]

 

Michaelis-Menten Model

substituting for the concentration of free enzyme and collecting all rate constants in one term gives the equation—>

 

where KM is called the Michaelis constant

[image]

KM is called the _____  _____.

Michaelis constant
 Michaelis-Menten Model

in the initial stages, formation of product depends only on the rate of breakdown of ES—represented by equa.—>

 Michaelis-Menten Model

if substrate concentration is so large that the enzyme is saturated with substrate [ES] = [E]T

equa.–>

[image]
 Michaelis-Menten Model

substituting k2[E]T = Vmax into the top equation gives  —>

[image]
 

when [S]= KM, the equation reduces to

[image]
Graphical determination of Vmax and KM from a plot of reaction velocity, V, against substrate concentration, [S].
[image]
 

Vmax is the constant rate reached when the enzyme is completely ______ with substrate, a value that frequently must be estimated from such a graph.

saturated
 Lineweaver-Burk Plot

which has the form y = mx + b, and is the formula for a straight line . equation?

[image]

 Lineweaver-Burk Plot

a plot of 1/V versus 1/[S] will give a ___ line with slope of KM/Vmax  and y intercept of 1/Vmax

such a plot is known as a Lineweaver-Burk ______  ______  ______.

straight

double reciprocal plot

Lineweaver-Burk Plot

 

a plot of 1/V versus 1/[S] will give a straight line with slope of _______and y intercept of ______ such a plot is known as a Lineweaver-Burk double reciprocal plot

KM/Vmax 

1/Vmax

 Lineweaver-Burk Plot

KM is the dissociation constant for ___ ; the greater the value of KM, the ____tightly S is bound to E

Vmax is the maximum ______

ES

less

velocity

 Lineweaver-Burk Plot

_____ is the dissociation constant for ES; the greater the value of ____ , the less tightly S is bound to E

 ____ is the maximum velocity

KM

KM

Vmax

 Lineweaver-Burk Plot

 

[image]

Turnover Numbers

Vmax is related to the turnover number of _____:also called kcat

 

enzyme

 Turnover Numbers

Vmax is related to the turnover number of enzyme:also called ______. 

 

kcat

 Turnover Numbers

Vmax is related to the turnover number of enzyme:also called kcat

 

[image]
 Turnover Numbers and KM

Values for some typical enzymes

[image]
 Enzyme Inhibition

Reversible inhibitor: a substance that binds to an ____  to ____ it, but can be

enzyme

inhibit

released

 Enzyme Inhibition

 

competitive inhibitor: binds to the ___ ____ site and blocks access to it by ____.

active (catalytic)

substrate

 Enzyme Inhibition

 

noncompetitive inhibitor: binds to a site other than the _____ _______ ; inhibits the enzyme by changing its ___.

 

active site

conformation

 Enzyme Inhibition

Irreversible inhibitor: a substance that causes inhibition that _____  _____ _____.

usually involves formation or breaking of  ___ ____ to or on the enzyme

cannot be reversed

covalent bonds

 

__________: a substance that binds to an enzyme to inhibit it, but can be released

Reversible inhibitor

 

__________: binds to the active (catalytic) site and blocks access to it by substrate

competitive inhibitor

 

___________: binds to a site other than the active site; inhibits the enzyme by changing its conformation

noncompetitive inhibitor
 

____________: a substance that causes inhibition that cannot be reversed

usually involves formation or breaking of covalent bonds to or on the enzyme

Irreversible inhibitor
 Competitive Inhibition

substrate must compete with inhibitor for the _____; more substrate is required to reach a given ________.

active site

reaction velocity

 Competitive Inhibition

substrate must compete with inhibitor for the active site; more substrate is required to reach a given reaction velocity

 

[image]
 Competitive Inhibition

substrate must compete with inhibitor for the active site; more substrate is required to reach a given reaction velocity

we can write a dissociation constant, KI for EI

[image]
look at me….
[image]
Structures of succinate, the substrate of succinate dehydrogenase (SDH), and malonate, the competitive inhibitor. Fumarate (the product of SDH action on succinate) is also shown.  
[image]

Competitive Inhibition

 

[image]
Competitive Inhibition
[image]
 Competitive Inhibition

 

 

In a Lineweaver-Burk double reciprocal plot of 1/V versus 1/[S], the  ______ (and the x intercept) changes but the y intercept does not _____.

 

slope

change

 Competitive Inhibition

[image]
 Noncompetitive Inhibition

 

 

several equilibria are involved…..

[image]
 The maximum velocity VImax has the form
[image]
Noncompetitivee inhibition
[image]
 Noncompetitive Inhibition

 

because the inhibitor does not interfere with binding of substrate to the active site, KM is ______.

increasing substrate concentration cannot overcome ______  _______.

unchanged

noncompetitive inhibition

Noncompetitive Inhibition
[image]
Noncompetitive Inhibition
[image]
Noncompetitive Inhibition
[image]

 Inhibitor Type

Competitive Inhibitor

Specifically at the catalytic site, where it competes with ______ for binding in a dynamic equilibrium- like process. Inhibition is  ______ by substrate.

 substrate

reversible

 

 Inhibitor Type

Competitive Inhibitor

Kinetic effect

 

Vmax is _______; Km, as defined by [S] required for 1/2 maximal activity, is increased.

unchanged

 Inhibitor Type

Noncompetitive Inhibitor

 

Binds E or ES complex other than at the _____  site. Substrate binding ______, but ESI complex cannot form products. Inhibition ____ _____ reversed by substrate.

catalytic

unaltered

cannot be

 Inhibitor Type

Noncompetitive Inhibitor

 

 

Km appears unaltered; Vmax is ________ proportionately to inhibitor _____________.

decreased

concentration

 Other Types of Enzyme Inhibition

 

Uncompetitive– inhibitor _____ bind to the ES complex but not to free _____.   Vmax decreases and KM decreases.

 

 

Mixed– Similar to noncompetitively, but binding of I affects binding of S and vice versa.

 

can

E

 Enzymes with Non-Michaelis Kinetics – Allosteric

Sigmoidal shape- characteristic of allosterism

Again Max. velocity reached, but different mechanism

[image]
 What Factors Influence Enzymatic Activity

 

 

Enzymes are highly regulated at

the activity level

 

Substrate-level Control; Product inhibition

 

What Factors Influence Enzymatic Activity

Rate slows as product ________.

 

Rate depends on ______  availability

 

________ effectors may be important

 

 

accumulates

substrate

Allosteric

Feedback Control (inhibition)

 

 

The protein level

Enzymes can be modified _______: reversibly or irreversibly

Zymogens, isozymes and modulator proteins may play a role

 

covalently

Translation – to make more or less ______.

Protein turnover

Compartmentalization

 

the gene level

Genetic controls – induction and repression

 protein

 

[image]
understand me
 Allosteric Enzymes

Allosteric: Greek allo + steric, other shape

Allosteric enzyme: an _____ whose biological activity is affected by other ______ binding to it.

these substances change the enzyme’s activity by altering the conformation(s) of its _____.

oligomer

substances

4° structure

 Allosteric effector: a substance that modifies the behavior of an  _________; may be an

allosteric ______ or an allosteric _______.

allosteric enzyme

inhibitor

activator

 Aspartate transcarbamoylase (ATCase)

   __________    _____________.

feedback inhibition
[image]
 ATCase

Figure à Rate of ATCase catalysis vs substrate conc.

[image]
 ATCase

Figureà ATCase catalysis in presence of CTP; ATP

[image]
 ATCase

catalytic unit:  ___ subunits organized into  trimers ___

regulatory unit: 6 subunits organized into 3 trimers

6

3

 Allosteric Enzymes

The key to allosteric behavior is the existence of multiple forms for the ___ ___ of the enzyme.

4° structure

 Allosteric Enzymes

 

allosteric effector: a substance that modifies the ____ of an allosteric enzyme

4° structure

 Allosteric Enzymes

 

homotropic effects: allosteric interactions that occur when several ____ molecules are bound to the ____; e.g., the binding of ____  to _____

identical

protein

aspartate

ATCase

  Allosteric Enzymes

 

heterotropic effects: allosteric interactions that occur when ______ substances are bound to the ______ ; e.g., inhibition of ATCase by ____  and activation by ______.

 

different

protein

CTP

ATP

 Allosteric Enzymes

 

________: a substance that modifies the 4° structure of an allosteric enzyme

allosteric effector
 Allosteric Enzymes

__________: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase

homotropic effects

 Allosteric Enzymes

________: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP

 

heterotropic effects
 General Features of Allosteric Regulation

Action at "another site"

Enzymes situated at key steps in ______ are modulated by

 

_______effectors.

metabolic pathways

allosteric

 General Features of Allosteric Regulation

Action at "another site"

 

These effectors are usually produced _____ in the pathway

 

elsewhere
 General Features of Allosteric Regulation

Action at "another site"

 

Effectors may be feed-forward ______ or feedback ____.

 

 

Kinetics are ____ ("S-shaped")

activators

inhibitors

sigmoid

 {Sigmoid v (allosteric)} versus { [S] plot (non competitive inhibition) }. The dotted line represents the hyperbolic plot characteristic of normal MichaelisMenten-type enzyme kinetics.
[image]
 Models for Allosteric enzymes

The ______ Model

 

The _______ Model

Concerted

Sequential

 The Concerted Model

Wyman, Monod, and Changeux – 1965

 

The enzyme has two conformations

R (relaxed): binds _____  tightly; the active form

substrate
 The Concerted Model

T (tight or taut): binds _____ less tightly; the inactive form

substrate
 The Concerted Model

 

In the absence of substrate, most enzyme molecules are in the  _______ form.

T (inactive)

 The Concerted Model

 

The presence of ____ shifts the equilibrium from the T (inactive) form to the R (active) form

substrate

 The Concerted Model

 

In changing from T to R and vice versa, all subunits change _________ simultaneously; all changes are concerted

conformation
 The Concerted Model

Wyman, Monod, and Changeux – 1965

The enzyme has two conformations

________: binds substrate tightly; the active form

________: binds substrate less tightly; the inactive form

 

R (relaxed)

T (tight or taut)

 Concerted Model

A ______ protein with two subunits

both change from ___ to ___ at the same time

hypothetical

T

R

 Concerted Model

Figureà Monod-Wyman-Changeaux model

[image]
[image]
  Sequential Model

Koshland – 1966

the binding of substrate induces a conformational change from the ___ form to the ____ form

T

R

 Sequential Model

Koshland – 1966

the change in conformation is induced by the fit of the  ______ to the enzyme, as per the induced-fit model of substrate binding

 

substrate
 Sequential Model

Koshland – 1966

the change in conformation is induced by the fit of the substrate to the enzyme, as per the _____ of substrate binding

 

induced-fit model
 Sequential Model

Figureà Sequential model for cooperative binding of substrate to an allosteric enzyme

[image]

 Sequential Model

Figureà Allosteric activation and inhibition also occur by the induced-fit mechanism

[image]
 Enzymes can be modified covalently

 

_______ Modification

_______ Modification

Reversible

Irreversible

 Phosphorylation

the side chain -OH groups of Ser, Thr, and Tyr can form  _______ esters

phosphorylation by ATP can convert an inactive precursor into an ______ ______.

phosphate

active enzyme

 

Irreversible Covalent Modifications:

Are known as ______

 

 

Zymogens

 Zymogens

Zymogen: an _____ precursor of an ______; cleavage of one or more _____  ______ transforms it into the active enzyme

 

inactive

enzyme

covalent bonds

 Zymogens

 Chymotrypsinogen

synthesized and stored in the _____

 a single polypeptide chain of 245 amino acid residues cross linked by five disulfide (-S-S-) bonds

pancreas
 Zymogens

when secreted into the small intestine, the digestive enzyme ______ cleaves a 15 unit polypeptide from the N-terminal end to give pchymotrypsin

trypsin
 Zymogens

_________: an inactive precursor of an enzyme; cleavage of one or more covalent bonds transforms it into the active enzyme

Zymogen
 The Active Site

1. Which amino acid residues on an enzyme are in the ____ _____  and  ____ the reaction?

active site

catalyze

 The Active Site

2. What is the spatial relationship of the _______ ______ _______ residues in the active site?

essential amino acids
 The Active Site

3. What is the _________ by which the essential amino acid residues catalyze the reaction?

 

mechanism

 The Active Site

As a model, we consider chymotrypsin, an enzyme of the digestive system that catalyzes the selective hydrolysis of peptide bonds in which the carboxyl group is contributed by Phe or Tyr

 

 The Serine Proteases

Trypsin, chymotrypsin, elastase, thrombin,  

subtilisin, plasmin, TPA

Enzyme and substrate become linked in a ______ ______at

 

one or more points in the reaction pathway

covalent bond
 The Serine Proteases

Trypsin, chymotrypsin, elastase, thrombin,  

 

subtilisin, plasmin, TPA

The formation of the ________ ______provides chemistry that

 

speeds the reaction

covalent bond
 The Serine Proteases

Trypsin, chymotrypsin, elastase, thrombin,  

 

subtilisin, plasmin, TPA

All involve a _______ in catalysis – thus the name

 

serine
 The Serine Proteases

Trypsin, chymotrypsin, elastase, thrombin,  

subtilisin, plasmin, TPA

 

Ser is part of a "catalytic triad" of _____, _____, ______.

Ser, His, Asp
 The Serine Proteases

Trypsin, chymotrypsin, elastase, thrombin,  

subtilisin, plasmin, TPA

Serine proteases are _____ , but locations of the three

crucial residues differ somewhat

homologous
 The Serine Proteases

Trypsin, chymotrypsin, elastase, thrombin,  

 

subtilisin, plasmin, TPA

Enzymologists agree, however, to number them always as His-

57, Asp-102, Ser-195

 

Burst kinetics yield a hint of how they work!

 

Chymotrypsin

because Ser-195 and His-57 are required for activity, they must be close to each other in the active site

results of x-ray crystallography show the definite arrangement of amino acids at the active site

in addition to His-57 and Ser-195, Asp-102 is also involved in catalysis at the active site

 Coenzymes

Coenzyme: _____ organic molecule that takes part in an _____ reaction and is regenerated for further reaction

 

 

nonprotein

enzymatic

 Coenzymes

organic compounds, many of which are vitamins or are metabolically related to vitamins are ______ – molecules that bring unusual chemistry to the enzyme active site

 

coenzymes
 Vitamins

Vitamins and coenzymes are classified as "___ ____" and "____ _____"

 

The water-soluble coenzymes exhibit the most interesting chemistry

 

water-soluble

fat-soluble

 Vitamins

Fat-soluble Vitamins:_____,_____,_____,_____.

 

A, D, E, K
 Vitamins

Water-soluble Vitamins:

  B1 – Thiamine  C – Ascorbic acid

  B2 –  Riboflavin 

  B3 –  Niacin

  B5 –  Pantothenic Acid

  B6 –  Pyridoxal

  B7 – Biotin

  B9 –  Folic acid

  B12 – Cyanocobalamin

 Nicotinic Acid and the Nicotinamide Coenzymes

aka pyridine nucleotides

These coenzymes are _____ _____ ______

 

two-electron carriers

Nicotinic Acid and the Nicotinamide Coenzymes

They transfer _____ ____ ____  to and from substrates

hydride anion (H)
 

Nicotinic Acid and the Nicotinamide Coenzymes

Two important coenzymes in this class:

 

Nicotinamide adenine dinucleotide ________

 

Nicotinamide adenine dinucleotide phosphate ______

 

(NAD+)

(NADP+)

 NAD+/NADH

NAD+ is a ____ ____ oxidizing agent, and is reduced to ______.

two-electron

NADH

 NAD+/NADH

NAD+ is involved in a variety of _____ ____ oxidation/reduction reactions.

enzyme-catalyzed
x

Hi!
I'm Larry

Hi there, would you like to get such a paper? How about receiving a customized one?

Check it out