Lecture 8 Enzyme

• Composition, structure and

properties of enzyme

• Enzyme kinetics

• Catalytic mechanisms of enzyme

• Regulation of enzyme activities

Outline

(1). Much of the early history of biochemistry is the history of enzyme research

(2). Biological catalysts were first recognized in studying animal food digestion and sugar fermentation with yeast (brewing and wine making)

(3). Ferments (i.e., enzymes, meaning in “in yeast”) were thought (wrongly) to be inseparable from living yeast cells for quite some time (Louis Pasteur)

1. Introduction to enzymes

(4). Yeast extracts were found to be able to ferment sugar to alcohol (Eduard Buchner, 1897, who won the Nobel Prize in Chemistry in 1907 for this discovery)

(5). Enzymes were found to be proteins (1920s to 1930s, James Sumner on urease and catalase ,“all enzymes are proteins”, John Northrop on pepsin and trypsin, both shared the 1946 Nobel Prize in Chemistry)

(6). Catalytic RNA (also called ribozyme ---from ribonucleic acid enzyme, or RNA enzyme) were found in the 1980s (Thomas Cech, Nobel Prize in Chemistry in 1989)

1. Definition of enzyme

•Enzymes are biological catalysts.•A Catalyst is defined as "a substance that increases the rate of a chemical reaction without being itself changed in the process.”

What is the difference between an enzyme and a protein?

Protein

•All enzymes are proteins except some RNAs • not all proteins are enzymes

RNAEnzymes

Enzymes are the most remarkable and specialized

biological catalysts• An enzyme catalyzes a chemical reaction at a

specifically structured active site, being often a pocket.

• Enzymes have extraordinary catalytic power, often far greater than those non-biological catalysts.

• Enzymes often have a high degree of specificity for their substrates.

• Enzymes are often regulatory.• Enzymes usually work under very mild conditions

of temperature and pH.• The substance acted on by an enzyme is called a

substrate, which binds to the active site of an enzyme in a complementary manner.

2. How enzymes work (important!)

1) Enzymes lower a reaction’s activation energy– All chemical reactions h

ave an energy barrier, called the activation energy, separating the reactants and the products.

– activation energy: amount of energy needed to disrupt stable molecule so that reaction can take place.

Enzymes Lower a Reaction’s Activation Energy

2) The active site of the enzyme • Enzymes bind substrates to their active site

and stabilize the transition state of the reaction.

• The active site of the enzyme is the place where the substrate binds and at which catalysis occurs.

• The active site binds the substrate, forming an enzyme-substrate(ES) complex.

Active siteBinding site

Catalytic site

Enzymatic reaction steps

1. Substrate approaches active site2. Enzyme-substrate complex forms3. Substrate transformed into products4. Products released5. Enzyme recycled

Characteristics of active sites

• The active site takes up a small part of the total volume of the enzyme.

• The active site is 3-dimensional and is generally found in a crevice or cleft on the enzyme.

• The active site displays highly specific substrate binding.

Active center of lysozyme129 aa, discovered by Fleming in 1922 in tears

Active center may include distant residues

3. Properties of enzymes (important!)

•Catalytic efficiency – high efficiency, 103 to 1017 faster than the corresponding uncatalyzed reactions

•Specificity - high specificity, interacting with one or a few specific substrates and catalyzing only one type of chemical reaction.

•Mild reaction conditions- 37℃ , physiological pH, ambient atmospheric pressure

High specificity

1). Absolute specificity: the enzyme will catalyze only one reaction.

e.g C O

NH2

NH2

H2O

脲酶CO2 + 2NH3

Urease

C O

NH2

NHCH3

H2O

脲酶X

Urease

(i) Group specificity: the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups.

2). Relative specificity

A — B or A — B

e.g α-D-glucosidase

O

CH2OH

OH

OH

OOH

R

(ii) Bond specificity: the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure.

3). Stereospecificity: the enzyme will act on a particular steric or optical isomer.

R1CO

OR2

+ H2O酯酶

R1COOH + R2OHesterase

C

C

H

HHOOC

COOH+ H2O

延胡索酸水化酶 CH2COOH

CHOHCOOH

fumarate hydratase

malate

4). Hypotheses of enzyme specificity(i) Lock and Key model

Proposed by Fischer in 1894

In this model, the active sites of the unbound enzyme is complementary in

shape to the substrate

(ii) Induced-fit modelProposed by Koshland in 1958

In this model, the enzyme changes shape on substrate binding

An Example: Induced conformational change in hexokinase

An Example: Induced conformational change in hexokinase

•Catalyzes phosphorylation of glucose to glucose 6-phosphate during glycolysis•such a large change in a protein’s conformation is not unusual

Conclusion

• Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate.

• The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction.

• Not a lock & key but induced fit – the enzyme and/or the substrate distort towards the transition state.

4 Chemical composition of enzymes

(1) Simple protein

(2) Conjugated protein

Holoenzyme= Apoenzyme+ Cofactor

Coenzyme : loosely bound to enzyme (non-covalently bound).

Prosthetic group : very tightly or even covalently bound to enzyme (covalently bound)

Cofactor

• Cofactors often function as transient carriers of specific (functional) groups during catalysis.

• Many vitamins and organic nutrients required in small amounts in the diet, are precursors of cofactors.

5 Classification of enzymes

1). Monomeric enzyme2). Oligomeric enzyme3). Multienzyme complex: such as Fatty acid synthase

(1). By their composition

(2) Nomenclature • Recommended name

•Enzymes are usually named according to the reaction they carry out.

•To generate the name of an enzyme, the suffix -ase is added to the name of its substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA polymerase forms DNA polymers).

•Systematic name (International classification)• By the reactions they catalyze (Six classes)

Transfer electrons (hydride ions or H atoms);play a major role in energy metabolism.

i.e., the transfer of functional groups to water.

e.g., the transfer of a phosphoryl group from ATP to many different acceptors.

These are direct bond breaking reactions without being attacked by

another reactant such as H2O.

Leading to the formation of C-C, C-S,C-O, C-N bonds.

Lactate dehydrogenase

NMP kinase

Chymotrypsin

Fumarase

Triose phosphateisomerase

Aminoacyl-tRNA synthetase

Each enzyme is given a systematic name and a unique 4-digit identification number for identification by the Enzyme Commission (E.C.) of IUBMB (since 1964)

Lactate dehydrogenase (lactate:NAD+ oxidoreductase)

lactate + NAD+ pyruvate + NADH + H+

1

Indicatestype of substrate

Indicatestype ofcofactor

6 Catalytic mechanisms of enzymes

• Mechanisms - the molecular details of catalyzed reactions

• How do enzymes stabilize the transition state of a reaction– General Acid-base catalysis– Covalent catalysis– Catalysis by proximity and orientation– Metal catalysis

1). General acid-base catalysis

• The active sites of some enzymes contain amino acid functional groups that can participate in the catalytic process as proton donors or proton acceptors --general acid-base catalysis.

•A general acid (BH+) can donate protons

•A covalent bond may break more easily if one of its atoms is protonated

Amino acids in general acid-base catalysis

2). Covalent catalysis• Covalent catalysis involves the substrate forming

a transient covalent bond with residues in the active site of the enzyme or with a cofactor.

• This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction.

• Group X can be transferred from A-X to B in two steps via the covalent ES complex X-E

A-X + E X-E + A

X-E + B B-X + E

•Examples of covalent bond formation between enzyme and substrate.

•In each case, a nucleophilic center (X:) on an enzyme attacks an electrophilic center on a substrate.

nucleophilic center (X:)

Nucleophilic group (X:)

Electrophilic group

3). Catalysis by proximity and orientation

• This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them close together.

•Analogous to an effective increase in concentration of the reactants.

4).Many enzymes have metal ions in their active centers playing important roles in catalysis.– help activate substrates– Stabilize charged transition

states by forming ionic bonds.

Substrate

Enzyme

•An enzyme may use a combination of several catalytic strategies to bring about a rate enhancement.

7. Enzyme activity• Enzymes are never expressed in terms o

f their concentration (as mg or μg etc.), but are expressed only as activities.

• Enzyme activity = moles of substrate converted to product per unit time.– The rate of appearance of product or the rat

e of disappearance of substrate– Test the absorbance: spectrophotometer

Units of enzyme activity:

• Katal (kat) – 1 kat denotes the conversion of 1 mole substrate per second (mol/sec)

• International unit (IU) - amount of enzyme activity that catalyses the conversion of 1 micromol of substrate per minute (μmol/min).

8. Factors affecting enzyme activity

• Concentration of substrate• Concentration of enzyme• Temperature• pH• Activators• Inhibitors

Enzyme velocity• Enzyme activity is commonly expressed

by the intial rate (V0) of the reaction being catalyzed. (why?)

• Enzyme activity = moles of substrate converted to product per unit time.

• Velocity decreases as time increases as:– S may be used up– P may inhibit reaction(E)– Change of pH may occur and decrease enzy

me reaction– Cofactor or coenzyme may be used up– Enzyme may loss activity

•At relatively low concentrations of substrate, Vo increases almost linearly (A) with an increase in [S].•At higher [S], Vo increases by smaller and smaller amounts (B) in response to the increase in [S].• Finally, a point (a plateau of maximum velocity, Vmax) (C) is reached.

•[S] is a key factor affecting the rate.

(1). Substrate concentration ([S]) affects the catalytic velocity (rate)

A

BC

Quantitative expression of relationship between [S] and V0

•1913, Leonor Michaelis and Maud Menten deduced the equation, Michaelis-Menten equation, based on the exist of intermediate ([ES]) in the enzyme reaction.

Leonor Michaelis (1875-1949)

Maud Menten (1879-1960)

•When [S] >> [E], E is saturated with S.

• ES complex formed when specific substrates fit into the enzyme active site (at the beginning of reaction)

Proposed Model

E + S ES E + P k1

k2

k3

• k1,k2 and k3 represent the velocity constants for the respective reactions.

Note: V means V0

[S]

[S] + KM

V = Vmax

Km: Michaelis constantKm = (k2 + k3)/k1

Michaelis-Menten equation (very important!)

• Michaelis-Menten equation describes how reaction velocity (V) varies with substrate concentration [S].

• The following equation is obtained after suitable algebraic manipulation.

The equation fits the observed curve very well.•When [S] is very low (<<Km), then V = (Vmax/ Km)[S] or V is linearly dependent on [S]. •when [S] is very high (>>Km), then V = Vmax; that is, the V is independent of [S].

Zero order reaction

First order reaction

[S]

[S] + KmV = Vmax

Significances of Km

1) When [S] = Km,

Vmax [S] Vmax [S] Vmax

V = = = Km+[S] [S] + [S] 2

so, when V = 1/2 Vmax , Km = [S], the unit of Km is as [S]

2) For a specific substrate, Km is a constant for the enzyme.

3) Km can be a measure of the affinity of E for S. A low Km value indicates a strong affinity between E and S.

• The lower the value of Km, the stronger affinity between E and S

Significances of Vmax

When [S] is very much greater than Km, Vmax [S] Vmax [S] V= = = Vmax

Km+[S] [S]

• Vmax is a constant. • Vmax is the theoretical maximal rate of the r

eaction - but it is NEVER achieved in reality.

Measurement of Km and Vmax

(i) Plot of V vs [S] If [S]<<Km If [S]>>Km

If [S]=Km

•Using the Michaelis-Menten equation, the Vmax is an asymptote and can thus only be approximated and as a result, the Km, which is Vmax/2, can't be determined accurately.

(ii) The double-reciprocal --Lineweaver-Burk plot

Transform the Michaelis-Menten equation to

maxmax

m

V

1

]S[

1

V

K

v

1

The double-reciprocal Lineweaver-Burk plot is a linear transformation of the Michaelis-Menten plot (1/V vs 1/[S])

(y=ax+b)

[S]>>[E] V∝[E] •The initial rate of an enzy

me-catalyzed reaction is always proportionate to the concentration of enzyme.

•This property of enzyme is made use in determining the serum enzyme for the diagnosis of diseases.

(2) Effect of [E] on velocity

(3) Effect of temperature on velocity

Bell-shaped curve

•It is worth noting that the enzymes have been assigned optimal temperatures based on the laboratory work.

(4) Effect of pH value on velocity

• Each enzyme has an optimal pH or pH range (where the enzyme has maximal activity).• Requirements for the catalytic groups in the active site in appropriate ionization state is a common reason for this phenomenon.

•The pH optimum varies for different enzymes.•Most enzyme: neutral pH (6-8).

Bell-shaped curve

(5) Effect of activator on velocity

(i). Inorganic ions

• Metal ions ， such as Na+, K+, Mg2+, Ca2+, Cu2+, Z

n2+, Fe2+ et al• Anions: such as Cl-, Br-, I- 、 CN- et al

(ii). Organic

(iii). Proteins

• Reducing agents, such as Cys 、 GSH

•Enzyme activators are molecules that bind to enzymes and increase their activity.

(6) Inhibition of enzyme activities(very important!)

• Inhibitor: any molecule which acts directly on an enzyme to lower its catalytic rate is called an inhibitor.(not denaturation)

• Some enzyme inhibitors are normal body metabolites.

• Other may be foreign substances,such as drugs or toxins.

Significance of studying inhibition of enzyme

• Relationship of structure & function of enzyme

• Mechanisms of enzyme catalysis

• Design of new drugs

Inhibition

• Irreversible inhibition

• Reversible inhibition

– competitive inhibition

– non-competitive inhibition

– uncompetitive inhibition

1). Irreversible inhibition •An irreversible inhibitor

binds tightly, often covalently, to amino acid residues at the active site of the enzyme, inactivating the enzyme.

.

Enzyme

SO I

•Irreversible inhibition is different from irreversible enzyme inactivation. •Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying protein structure but by specifically altering the active site of their target.

• DFP is an organic phosphate that inactivates serine proteases, it can react with the active site serine (Ser-195) of enzyme to form DFP-E.• These inhibitors are toxic because they inhibit acetylcholin esterase (a serine protease that hydrolyzes the neurotransmitter acetylcholine). • Such organophosphorous inhibitors are used as insecticides or for enzyme research.

DFP

Examples: diisopropyl fluorophosphate( DFP)

• Many poisons are harmful to cells because they are potent irreversible inhibitors. Examples are heavy metals (mercury, lead, silver, ……)

• Heavy metals such as Ag+, Hg2+, Pb2+ have strong affinities for sulfhydral (-SH) groups.

• Since many enzymes contain -SH as part of their active sites, any chemical which can react with them acts as an irreversible inhibitor.

Heavy metals

Enzyme Enzyme

2). Reversible enzyme inhibition

•Inhibitor ( I ) binds to an enzyme and prevents formation of ES complex or breakdown to E + P.• Reversible inhibitors bind to the enzyme via noncovalent interactions and can be dissociated readily from the enzyme. • There are three basic types of inhibition: Competitive, Noncompetitive and Uncompetitive.

(i) Competitive inhibition

•Competitive inhibitors usually resemble the substrate

•Substrate cannot bind when I is bound at active site(S and I “compete” for the enzyme active site)

• Inhibitor binds only to free enzyme (E) not (ES)

• The effect of a competitive inhibitor can be overcomed with high concentrations of the substrate

Example 1 - competitive inhibition

• Malonate is a competitive inhibitor of succinate for succinate dehydrogenase

Clinical significance of enzyme inhibition

• The usefulness of the most important pharmaceutical agents, antimetabolites, is based on the concept of competitive enzyme inhibition.

• The antimetabolites are structural analogues of normal biochemical compounds.

• As competitive inhibitors they compete with the naturally substrate for the active site of enzyme and block the formation of undesirable metabolic products in the body.

• They are in use for cancer therapy, gout etc.

Example - Competitive Inhibition

• Sulfanilamides (also known as sulfa drugs, discovered in 1932) were the first effective systemic antibacterial agents.

• Sulfanilamide is a competitive inhibitor of p-aminobenzoic acid (PABA).

COOH

NH2

p-aminobenzoic acid

folic acid

SO2NH2

NH2

sulfanilamide

• p-aminobenzoic acid (PABA) is required by many bacteria to produce an important enzyme cofactor, THF. These bacteria require THF for their growth and division.

•Sulfanilamide acts as a competitive inhibitor to enzymes that convert PABA into folic acid, resulting in a depletion of this cofactor.

•The depletion of this cofactor results in retarded growth and eventual death of the bacteria.

• Mammals absorb their folic acid from their diets, so sulfanilamide exerts no effects on them.

Mammal: Folic acid (diet)

DHF THF

Bacteria: PABA DHF THF

Kinetics of competitive inhibition

Direct and Lineweaver-Burk plot

•Vmax does not change•At a sufficiently high [S], the reaction velocity reaches the Vmax observed in the absence of inhibitor.

•Km increases•This means that in the presence of a competitive inhibitor more substrate is needed to achive ½ Vmax.

Km Km’ [S], mM

II

voVmax

(ii) noncompetitive inhibition

•The inhibitor binds at a site other than the active site on the enzyme surface.

•This binding impairs the enzyme function.

•The inhibitor has no structural resemblance with the substrate.

•Inhibition cannot be overcome by addition of S.

Inhibitors bind to both E and ES

noncompetitive inhibition

•Vmax decreases•Noncompetitive inhibition cannot be overcome by increasing [S].

•Km does not change•Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme.

Kinetics of noncompetitive inhibition

Direct and Lineweaver-Burk plot

Km

Vmax

[S], mM

vo

IIVmax’

= Km’

(iii) Uncompetitive inhibition

•Inhibitors bind to ES not to free E.•The effect of an uncompetitive inhibitor can not be overcome by high concentrations of the substrate.

Kinetics of uncompetitive inhibition Direct and Lineweaver-Burk plot

Vmax decreasedKm also decreased

•Lines on double-reciprocal plots are parallel.

•This type of inhibition usually only occurs in multisubstrate reactions.

Km [S], mM

Vmax

II

Km’

Vmax’

V0

Enzyme Inhibition (summary)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] only,and competes with [S];increasing [S] overcomesInhibition by [II].

[II] binds to free [E] or [ES] complex; Increasing [S] cannot overcome [II] inhibition.

[II] binds to [ES] complex only, increasing [S] favorsthe inhibition by [II].

E + S → ES → E + P + II↓EII

←

↑

E + S → ES → E + P + + II II↓ ↓EII + S →EIIS

←

↑ ↑

E + S → ES → E + P + II ↓ EIIS

←

↑

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ec

t P

lots

Do

ub

le R

eci

pro

cal

Vmax Vmax

Km Km’ [S], mM

vo

[S], mM

vo

II II

Km [S], mM

Vmax

II

Km’

Vmax’Vmax’

Vmax UnchangedKm Increased

Vmax DecreasedKm Unchanged

Both Vmax & Km Decreased

II

1/[S]1/Km

1/vo

1/ Vmax

II

Two parallellines

II

Intersect at X axis

1/vo

1/ Vmax

1/[S]1/Km 1/[S]1/Km

1/ Vmax

1/vo

Intersect at Y axis

= Km’

9. Regulation of enzyme activities

•The activity of some enzymes are precisely and tightly regulated in living organisms to meet physiological requirements.

•Regulatory enzymes - activity can be reversibly modulated by effectors.

•Such enzymes are usually found at the first unique step in a metabolic pathway.

•Regulation at this step conserves material and energy and prevents accumulation of intermediates.

Regulation of enzyme activity

• Allosteric enzyme• Reversible covalent modification• Proteolytic activation

(1). Allosteric regulation

•Allosteric enzymes have a second regulatory site (allosteric site, Greek: allo-other) distinct from the active site.

•Allosteric inhibitors or activators bind to this site (noncovalently) and regulate enzyme activity via conformational changes.

•Regulatory enzymes possess quaternary structure (contain multiple subunits).

•There is a rapid transition between the active (R) and inactive (T) conformations

R

T

• There is a rapid transition between the active (R) and inactive (T) conformations.

• Substrates and activators may bind only to the R state while inhibitors may bind only to the T state.

sigmoidal-shaped curve

• A plot of V0 against [S] for an allosteric enzyme gives a sigmoidal-shaped curve (normal enzyme is a hyperbolic curve).

(2). Reversible covalent modification1). What’s covalently modulated enzymes?•Activity is modulated by covalent modification of one or more of its amino acid residues in the enzyme molecule.

Enzyme

Modifyinggroup

EnzymeModifyinggroup

Inactive Enzyme Active Enzyme

• Common modifying groups include: phosphoryl, adenylyl, methyl, hydroxyl, sulfate and ADP-ribosyl groups.

• These groups are generally linked to and removed from the regulatory enzyme by separate enzymes.

Phosphorylation and dephosphorylation

• The most common such modification is the addition and removal of a phosphate group: phosphorylation and dephosphorylation, respectively.

• Phosphorylation is catalyzed by protein kinases, often using ATP as the phosphate donor.

• Dephosphorylation is catalyzed by protein phosphatases.

Protein kinases catalyze the phosphorylation of proteins

OH

++

PO-

O

O-

++

ATP ADP

Tyrosine Kinase

O

CH2OH

++

PO-

OO

+ +

CH2

ATP ADP

Serine/ThreonineProtein Kinase O-

Protein phosphatases remove phosphate groups from phosphorylated proteins

OH

++

+ +

O P O-O-

O

ATP ADP

Pi

Protein Kinase

Protein Phosphatase

•Phosphorylation and dephosphorylation are not the reverse of one another.• The rate of cycling between the phosphorylated and the dephosphorylated states depends on the relative activities of kinases and phosphatases.

Response of enzyme to phosphorylation

• Depending on the specific enzyme,the phosphorylated form may be more or less active than the unphosphorylated enzyme.

• For example,phosphorylation of glycogen phosphorylase (GP,an enzyme that degrades glycogen) increases activity, whereas the addition of phosphate to glycogen synthase (GS,an enzyme that synthesizes glycogen) decrease activity.

(active form)

(active form)

(inactive form)

(inactive form)

Phosphorylation Is a Highly Effective Means of Regulating the Activities of Target Proteins

Inactive protein kinase 1

Inactive protein kinase 2

Inactive protein kinase 3

Inactive protein kinase 4

Inactive protein kinase 5

Inactive protein kinase 1

Phoshporylation cascade Cellular response

Active proteinPhosphatase

• Some enzymes are synthesized as larger inactive precursors calles proenzymes or zymogens.

• These are activated by the irreversible hydrolysis of one or more peptide bonds.

• The pancreatic proteases trypsin, chymotrypsin and elastase are all derived from zymogen precursors by proteolytic activation.

(3). Zymogen or proenzyme

The central role of trypsin: it is the common activator of all

pancreatic zymogens.

autocatalysis in the small intestine

cascade

Activation of chymotrypsinogen to chymotrypsin, and of trypsinogen to trypsin

Acute pancreatitis• Premature activation of these zymogens lead

s to the condition of acute pancreatitis.– The exocrine pancreas produces a variety of enzy

mes, such as proteases, lipases, and saccharidases.

– These enzymes contribute to food digestion. – In acute pancreatitis, the worst offender among t

hese enzymes may well be the protease trypsinogen which converts to the active trypsin which is most responsible for auto-digestion of the pancreas which causes the pain and complications of pancreatitis.

10. Isoenzyme or isozyme

Definition: • Enzymes in an organism that catalyze the same reaction but differ in structure; these differences may range from one to several amino acid residues.

Lactate dehydrogenase (LDH)

• LDH is a tetramer of two different types of subunits,called H and M, which have small differences in amino acid sequence.•The two subunits can combine randomly with each other,forming 5 isoenzymes that have the compositions H4, H3M, H2M2, HM3, M4.

HHHH

MHHH

MMHH

MMMM

MH Heart type Muscle type

MMMH

O O-

CH

C

CH3

HO

L-lactate

O O-

C

C

CH3

O

pyruvate

NADH+ H+NAD+

NADH+ H+NAD+

11. Enzymes in clinical diagnosis

• An enzyme test is a blood test or urine test that measures levels of certain enzymes to assess how well the body’s systems are functioning and whether there has been any tissue damage. (why?)

Plasma enzymes• Plasma enzymes can be classified into two

major groups:1) a relatively small group of enzymes are actively

secreted into the plasma by certain organs. • For example,the enzymes involved in blood coagulatio

n.2) a large number of enzyme species are released f

rom cells during normal cell turnover.These enzymes are normally intracellular and have no physiologic function in the plasma.

• In healthy individuals, the levels of these enzymes are fairly constant.

• The presence of elevated enzyme activity in the plasma may indicate tissue damage.

• Common enzymes used for clinical diagnosis include:– alanine aminotransferase(ALT,also called

glutamate pyruvate transaminase,GPT) – alkaline phosphatase – amylase – aspartate aminotransferase – creatine kinase – lactate dehydrogenase

Points• How enzymes work

– lower a reaction’s activation energy, active site • Properties of enzymes

– highly efficiency, highly specific, mild reaction conditions,

– Hypothese of enzyme specificity: induced-fit model• Classification of enzymes: six classes• Enzyme kinetics

– Substrate Concentration: Michaelis-Menten equation, Km, Vmax, double-reciprocal plot

– Inhibition of enzyme activities: Irreversible inhibition, Reversible inhibition (competitive, non-competitive, uncompetitive inhibition)

• Regulation of enzyme activities– Allosteric enzyme, Reversible covalent modification,

Proteolytic activation• Isoenzyme