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Flashcards in Enzyme Kinetics Deck (48):

1. What are they?
2. Rate of rxn and equilibrium
3. Energy barrier?
4. K eq and Delta G

Are catalysts
Increase the rate of a reaction, but do not effect the equilibrium.
Decrease the energy barrier of the reaction.
Enzyme do not effect the Delta G of the reaction, and thus does not effect the K eq.


Four enzyme characteristics
1. Rxn rate
2. Conditions?
3. Specificity?
4. Regulation?

1. Enzymes result in higher rxn rates: 10^6-10^12 greater than non-catalyzed rxn's, and at least 10^3 greater than non-enzymatic catalyze rxn's.
2. Milder reaction conditions: Enzymatic rxn's occur at atmospheric pressure, neutral pH, and temperatures less than 100 C.
3. Tremendous reaction specificity: Specificity results in very rarely occurring side products.
4. Enzymes can be regulated: enzyme activity can vary in response to biological molecules other than the enzymes substrates or products.


Measurement of enzyme activity
1. Enzyme activities are expressed as Units
2. Specific activity

1. A unit of enzyme activity is the amount (usually micromoles) of substance converted to product in a given amount of time (usually minutes) i.e. One unit= 1micromole/min.
2. The specific activity of an enzyme is the number of units per mg protein.


Effects of temperature and pH on enzymes

Temperature: enzymes have a temperature optimum. Most human enzymes have a temperature optimum of about 37*C.
pH: enzymes have a pH optimum. Pepsin (stomach enzyme) has a pH optimum of 2, Trypsin (small intestine enzyme) has pH of 8



A. Catalyze oxidation-reduction reactions
B. Dehydrogenases, oxidases, reductases, peroxidases, catalase, oxygenases, hydroxylyases



A. Catalyze transfer of a group such as glycosyl, methyl, or phosphoryl
B. Transaldolase, transketolase, acyl, methyl, glucosyl, and phosphoryl transferase, kinases, phosphomutases
C. Transfer C, N, P containing group



A. Catalyze hydrolytic cleavage of C-C, C-O, C-N and other bonds.
B. Easterases, glycosidases, peptidases, phosphatases, etc.



A. Catalyze cleavage of C-C, C-O, C-N and other bonds by atom elimination, leaving double bonds.
B. Decarboxylases, aldolases, hydratases, dehydratases, synthases.



A. Catalyze geometric or structural changes within a molecule.
B. Racemases, epimerases, isomerases, some mutases.



A. Catalyze the joining together of two molecules coupled to the hydrolysis of ATP.
B. Synthetases and carboxylases


Active site
1. Where?
2. Specificity?
3. Structure of enzyme?
4. Residue?
5. Changes?

1. A specific region on the enzyme where the catalysis occurs.
2. Substrate binds to the active site along with any other required elements in a highly specific manner which promotes the reaction which is being catalyze.
3. The structure of the enzyme (protein) is critical for the appropriate structure of the active site.
4. There may be residues which play an important role in the catalyze reaction (ex. Serine proteases require a serine residue at the active site)
5. Changes in the enzyme structure (due to changes in the amino acid composition of the enzyme) may or may not have major effects on the activity of the enzyme.


Catalysis by proximity

1. For molecules to react they must come within bond forming distance of each other.
2. The active site of an enzyme binds substrate(s) creating a high local concentration.
3. The substrates are also bound in a specific orientation conductive to reaction.


Acid-base catalysis

Ionizable functional groups of amino acyl side chains may participate in the catalysis by acting as acids and/or bases.


Catalysis by strain

Enzymes which catalyze reactions which break bonds may bind substrate(s) in such a way to destabilize the bond to be broken.


Covalent catalysis

1. A covalent bond is established between a substrate and the enzyme.
2. The covalently modified enzyme essentially then becomes a substrate for a subsequent reaction (to produce the ultimate product).
3. The reaction of covalently modified enzyme to product is energetically more favorable than the reaction of substrate to product.


Features of cofactors, coenzymes, and prosthetic groups

1. Many enzymes require these.
2. Small no-protein molecules and metal ions which participate directly in the enzymatic process.
3. Many are derived from vitamins.


Prosthetic groups

Tightly and stably incorporated into the protein, sometimes by covalent bonds.
1. Metal ions are the most common prosthetic groups (Co, Cu, Mg, Mn, and Zn)
2. Enzymes containing a metal prosthetic group are called metalloenzymes.
3. Some other examples include:
-pyridoxal phosphate (derived from the vitamin pyridoxine, B6)
-thiamine pyrophosphate (derived from thiamine, B1)
-Lipoic acid
-Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD, derived from riboflavin, B2)



Bind transiently to either the substrate or the enzyme, but are nonetheless required for activity.
-The most common example is Mg++, required for enzymes which involve ATP.



Serve as "shuttles" or "transfer agents", accepting a group from on reaction, and supplying the group in other reactions.
-Coenzyme A (CoA, derived from pantothenic acid) transfers acyl sequences)
-Folates (several forms, derived from folic acid) transfers one carbon groups.


Isonzymes- definition

Physically distinct enzymes (i.e. Different proteins, with different amino acyl sequences) which catalyze the same reaction are called isoenzymes or isozymes.


Isozymes- Properties

-There may be kinetic differences, such as different substrate affinities.
-There may be differences in activity regulation.
-Isozymes may be differentials express by different tissues. ex. the heart isozymes of creatine kinase (CK) may be distinguished from the skeletal muscle isozymes of CK.


Velocity (v)

-measured by the disappearance of reactant (A) or by the appearance of product (P) per unit time, and is a function of the concentration of reactant:




Zero order reaction



First order reaction



Second order reaction




-When [S] is very high, the enzyme will be saturated with S such that [ES]=[E total]
-At this point, since the enzyme is saturated with S, adding more S will not result in an increase in activity, therefore, this represents the maximum velocity of Vmax


Km and affinity

-Km equals the S at which the velocity is one-half maximum
-Enzymes with lower Km, are said to have a greater affinity for their substrates than enzymes with higher Km
-a. A lower Km means a lower conc. of S is required to achieve 1/2 Vmax
-b. Since Km represents (k3+k2/k1), a low Km means formation of ES is more rapid than the breakdown of ES


k3 and kcat

-k3 is usually the rate limiting step is catalysis, and therefore may be called kcat, or the turnover number


Competitive inhibition

A. The inhibitor competes with the substrate for the active site of the enzyme.
1. The inhibitor usually has some structural similarity to the substrate.
2. The inhibitor binds only to the free enzyme.


Competitive inhibition and "apparent Km"

-inhibitor removes some of the free E from the above reaction, it appears to take a higher [S] to achieve 1/2 Vmax
-Vmax is unaffected, since by increasing [S], the inhibition can be overcome and Vmax achieved.
-results in an intersection on the y-axis
-Statins are competitive inhibitors of the rate limiting enzyme of cholesterol biosynthesis


Noncompetitive inhibitor

-binds to either E or ES
-do not bind with active site and therefore do not compete with substrate binding
-since inhibitor does not compete with the S, increasing [S] will not overcome the inhibition


Noncompetitive inhibitor and "apparent" Vmax

-the noncompetitive inhibitor reduces the number of active enzyme molecules, which has the appearance of reduction [Etotal]
-results in an intersection on the -x axis
-many heavy metals (Hg and Pb) interact with the -SH groups on enzymes, and act via noncompetitive inhibition.


Uncompetitive inhibition

-binds only to the ES of an enzyme
-propose that substrate binding alters the enzyme structure such that the inhibitor may subsequently bind
-does not compete with the substrate for the active site, and therefore the inhibition can not be overcome by an increase in [S]
-As [S] increases, so does inhibition since ES is required for the inhibitor, therefore Vmax is decreased.
-are rare in single substrate enzymes, but more common in multi-substrate enzymes


Uncompetitive inhibitor and Lineweaver-Burk plot

-both Km and Vmax are affected
- apparent Vmax less than the true Vmax, and an apparent Km less than the true Km
-results in a parallel plot


Allosteric enzymes

-display positive cooperatively with their substrate
1. Binding of one substrate molecule facilitates the binding of subsequent molecules to the enzyme.
2. Allosteric enzyme has more than one substrate binding (and active) site, and usually is composed of more than one subunit.


Hill number

-n is the Hill number
A. The Hill number is a measure of the degree of cooperativity.
B. The larger the value of n, the greater the cooperativity, and the greater the sigmoidicity of the curve
C. If n=1, there is no cooperativity, and the equation becomes the Michaelis-Menton equation


K 0.5

-not the same as Km
-K 0.5 does not represent the [S] at 1/2 maximal velocity
-since the rate constants for an allosteric enzyme are different than those for a Michaelis-Menton enzyme, the two constants are not equal


Conformational changes in allosteric enzymes

-my be in "tense" or "relaxed" confirmation
1. At low [S], the enzyme is in the "tense," T, inactive conformation
2. Binding of S promotes a conformational change to or toward the "Relaxed," R, active conformation of the enzyme


Homotropic regulation

-always positive
-binding of S to an allosteric enzyme increases the affinity of the other catalytic site for S


Heterotropic regulation

-regulatory molecule distinct from the substrate which binds to a site other than the catalytic site (other site) and modulates the activity of the enzyme


Negative and positive heterotropic effectors of allosteric enzymes

-heterotropic effectors interact with a site other than the active site (or substrate binding site) and change the kinetic properties of the enzyme
-effectors may be either negative (inhibitors) or positive (activators)


V-system effectors

1. May affect the catalytic rate, and thus influence the Vmax
2. The effect may be either positive or negative


K-system effectors

1. These effects affect the binding of substrate and thus influence the K 0.5.
2. The effect may be positive or negative.



-conflicting metabolic pathways are restricted to specific organelles
-enzymes, cofactors, and substrates of two conflicting pathways physically separate, futile cycling of substrates is avoided.
-Ex: fatty acid synthesis occurs in the cytoskeleton, fatty acid oxidation occurs in the mitochondrion.


Allosteric regulation of hormones

-produce second messengers which are allosteric effector (both negative and positive) of various enzymes in various tissues
1. Many hormones stimulate the production of cyclic AMP (cAMP) binds various enzymes, altering their activity
2. Some hormones increase the cytosolic [Ca++], which interacts with a protein called cal model in. The Ca+2-calmodulin complex affects the activity of a number of proteins.



-is accomplished by a protein kinase which may be very specific for the enzyme or protein to be phosphorylated or may be more broad in its specificity
-Dephosphorylation is accomplished by a protein phosphatase which may be very specific or relatively non-specific.


Zymogen (proenzyme)

Inactive from and proteolytic cleavage activates
-examples are important proteolytic enzymes involved in digestion and blood coagulation
A. Digestive proteases: trypsinogen, chymotrypsinogen
B. Coagulation zymogens: fibrinogen, prothrombin


Glycogen phosphorylase activity

-increased by the allosteric effector AMP
-increased by phosphorylation of the enzyme
-increase by the binding of Ca+2-calmodulin