1 enzymes. objectives what are enzymes ? properties of enzymes classification factors affecting...
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EnzymesEnzymes
ObjectivesObjectives
• What are enzymes ?
• Properties of enzymes
• Classification• Factors Affecting Enzyme Action• Enzyme Kinetics
What Are Enzymes?What Are Enzymes?
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• BiologicalBiological CatalystCatalyst
• Most enzymes are Proteins Proteins
• Not permanentlyNot permanently changed in the process
• Not consumedNot consumed
• Differ from inorganic catalysts in:
•Being sensitive to changes in pH , temperature ,
•loss of activity by time •specificity
Definitions•Ribozymes : are RNAs with catalytic activity.
•catalyze the cleavage & synthesis of phosphodiester bonds
•Zymogen•Isozymes
Isoenzymes:• enzymes that catalyze the same
reaction but may have genetically determined difference in the amino acid sequence
•Thus may have difference in their physical properties
The Active Site
• Plasma enzymes 2 major groupsmajor groups:
•1-Functional plasma enzymes• perform specific physiological function.• Examples:• Zymogens (inactive
precursors) of enzymes involved in blood coagulation.
•2-Non-functional plasma enzymes
• no physiological role in the blood. • occur in blood in very minute amounts).
• Enzymes composed wholly of protein are known as simple enzymes
• ComplexComplex enzymes, which are composed of protein + small molecule holoenzymes.
• Protein component apoenzyme, while the non-protein component coenzyme or cofactor
assistance
• Prosthetic group describes a complex in which the small organic molecule is permanently bound to the apoenzyme by covalent bonds and cosubstrate occurs when it is transiently attached.
• Coenzymes are often derivatives of vitamins (FAD,NAD, CoA)
• Catalytic efficiency:Typically 100-1000 substrate
molecules transformed to product / second ?(by enz.)
Turn over number : is the number of substrate moles converted to product/enzyme mol /sec.
Location within the cell:•Mitochondria: TCA cycleFatty oxidationDecarboxylation•Cytosol:GlycolysisHMPFatty acid synthesis•Lysosomes:Degradation of macromolcule
Nomenclature of Enzymes& Classification
•Recommended name uricase ,glucosidase
Systematic Name
•Currently enzymes are grouped into six functional classes by the International Union of Biochemistry & Molecular Biology (I.U.B.M.B
•Regulation:
Rate of product formation responds to the needs of the cells
Factors Affecting Enzyme Action
Temperature
Optimum temperature
ReactionRate
Low High Temperature
Substrate Concentration
Maximum activity
ReactionRate
substrate concentration
pH:
ReactionRate
Optimum pH
3 5 7 9 11
pH
Narrow range of activityMost lose activity in low or high pH
• This is because by time the substrate decreases (↓v1 ) ,
• the product increases(↑v-1 ) besides
• the enzyme decreases due to denaturation.
Time
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How do enzymes Work?
Enzymes work by
weakening bonds which
lowers activation
energy
Substrate
If enzyme just binds substrate then there will be no further reaction
Transition state Product
Enzyme not only recognizes substrate, but also induces the formation of transition state
X
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Enzymes
FreeEnergy
Progress of the reaction
Reactants
Products
Free energy of activationFree energy of activation
Without Enzyme
With Enzyme
The active site consists of certain groups e.g.-SH, -OH, COO-, NH3 + & imidazole.
Some groups are concerned with binding of the substrate & that determines the specificity of the enzyme.
• Other groups are concerned with catalysis.
• In some enzymes these groups can participate in general acid-base catalysis. In others, catalysis may involve transient formation of a covalent enzyme-substrate complex.
Inhibition of enzyme activity
• InhibitorInhibitor :is any substance that can diminish the velocity of the enzyme catalyzed reaction.
• Competitive
• Non competitive: Heavy metal ions (e.g. mercury and lead) should generally be prevented from coming into contact with enzymes as they usually cause such irreversible inhibition by binding strongly to the amino acid backbone.
• Uncompetitive
Reversible Inhibition:Competitive
Non Competitive Inhibition:
Enzyme Inhibition (Mechanism)
I
I
S
S
S I
I
I II
S
Competitive Non-competitive Uncompetitive
EE
Different siteCompete for
active siteInhibitor
Substrate
Ca
rtoo
n G
uid
eEq
uatio
n an
d De
scrip
tion
[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
Juang RH (2004) BCbasics
Sulfa Drug Is Competitive Inhibitor
-COOHH2N-
-SONH2H2N-
Pteridine PrecursorFolicacid
Tetrahydro-folic acid
SulfanilamideSulfa drug (anti-inflammation)
Para-aminobenzoic acid (PABA)
Bacteria needs PABA for the biosynthesis of folic acid
Sulfa drugs has similar structure with PABA, andinhibit bacteria growth.
Adapted from Bohinski (1987) Modern Concepts in Biochemistry (5e) p.197
Regulation of Enzyme Activity
•Substrate availability•Allosteric Effectors•Covalent modification•Induction & repression
• Regulation maybe short term regulation or long term regulation
• The long term regulation includes enzyme synthesis & repression
• The short term regulation includes allosteric regulation & covalent modification
Allosteric Effectors
•AllostericAllosteric means (“occupy another space”, other than that of the substrate).
•Allosteric enzymesAllosteric enzymes are enzymes whose activity at the catalytic site may be modulated by the presence of allosteric effectors at an allosteric site (i.e. at a site other than the active site.)
•Allosteric enzymes usually contain multiple subunitsusually contain multiple subunits.•The molecules regulating the allosteric enzymes are called effectors (modifiers or modulatorsmodifiers or modulators).
• Allosteric effectors bind non-covalentlynon-covalently at a site other than the active site.
• Binding of allosteric effectors at the allosteric site induces conformational changes at the catalytic site & this can alter the affinity of the enzyme for its substrate or modify the maximal catalytic activity of the enzyme or both.
Covalent Modification
•Addition or removal of phosphate groups from specific serine, threonine, tyrosine
•The phosphorylated form of the enzyme may be activated or deactivated according to the specific enzyme
Result of Regulation
Blood SampleBlood Sample
Plasma X Serum
Physiological Lab.
Thank You
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EnzymesEnzymes•Are specific
for what they will catalyzecatalyze
•Are ReusableReusable
• Enzymes as indicator tools
• In healthy individuals, levels of these enzymes are almost constant as it reflects a steady state
• elevated enzyme activity in plasma may indicate tissue damage which is accompanied by increased release of intracellular enzymes.
Enzyme Stabilizes Transition State
S
P
ES
EST
EP
ST
Reaction direction
Energy change
Energy required (no
catalysis)
Energy decreases (under
catalysis)
T = Transition state
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166