Summary
ENZYMES
What is the concept of catalysis? Include 4 catalytic mechanisms
catalysis = increase rate of reaction (both directions) without affecting
final equilibrium of reactants and products or dG eg. catalase 10^14x faster
to a Kcat of 40000000s-1
reduces activation energy of reaction (difference between free energy of
ground and transition state)
1. acid base catalysis - using proton from environment/substrate/active site
to stabilise unstable charged intermediates to lower Ea, make stronger
nucleophiles/electrophiles
2. covalent catalysis - forms transient bond between E and S to make
intermediate with lower activation pathway
3. bond strain - forcing substrate into conformation that strains bonds to inc
likelihood of transition state formation
4. metal ion catalysis - help orientate substrate, stabilise charged
intermediates, mediate redox
Definitions of transition state, isoenzymes, co factors, coenzymes,
intermediate
transition state = unstable, high energy, transient intermediate (highest
energy state is RDS), equally likely to decay to substrate and product
intermediate = compound formed along the pathway with a transient
lifetime eg. ES complex
isoenzymes = catalyse same reaction but different structure/region
(different gene coding and regulation), may have different pH/temp
optimums and Km/Vmax etc but same Keq
cofactors = non protein helper needed for function, inc organic co
enzymes and inorganic ions eg. Cu+ for cytochrome oxidase
Summary 1
, coenzymes = organic, called prosthetic group if tightly bound to enzyme,
transient carriers of atoms/functional groups eg. FAD (from riboflavin)
transfers electrons
What is the meaning of substrate specificity? Give three models to
demonstrate
specificity = enzymes only act on one type of reaction - one substrate or
class of similar molecules, one stereoisomer eg. alcohol dehydrogenase
oxidises primary alcohols to aldehydes
specific residues in active site are important - eg. glyceraldehdye and G3P
both reacted with triose phosphate isomerase but the phosphate group
shows 80% rate acceleration for G3P
1. lock and key (fisher) = shape of substrate is complementary to shape of
enzyme active site
2. induced fit (koshland) = shape of enzyme active site changes upon
binding (induced by weak interactions with substrate), bringing catalytic
residues into position eg. hexokinase binding glucose and MgATP
3. new model = active site is complementary to the transition state - releases
free binding energy when complex forms = stabilises, lowers Ea, reduces
entropy and desolvates substrate
(specific activity = purity of enzyme preparation ie. how many enzyme
units per amount of protein)
What are the 6 classes of enzymes and examples?
classes (HOTLIL)
1. hydrolases eg. trypsin splits Lys-Val - use H2O to split into two molecules
2. oxidoreductases eg. lactate dehydrogenase: pyruvate + NADH + H+ >
lactate + NAD+ (ie. transfer of hydride H- ion) redox transfer of H/O/e
3. transferases eg. alanine aminotransferase (glutamate + pyruvate <> L-
alanine + a-ketoglutarate) or hexokinase - transfer functional groups
between molecules
Summary 2
, 4. lyases eg. aldolase splits F16BP to DHAP and G3P, dopa decarboxylase -
split/join molecules by breaking covalent bonds
5. isomerases eg. phosphoglucose isomerase G6P>F6P - move atoms within
one molecule
6. ligases eg. DNA ligase - join two molecules by forming new covalent
bonds C-C/S/O/N and hydrolysing ATP
further classed by specific type of reaction to give 4 digit number
What is the effect of pH and temp on enzyme activity
pH and temp alter the bonds holding the enzyme in its specific shape,
destroying the active site and catalysis - different enzymes have different
optimums
pH change > (de)protonation of side chains of amines/carboxylic acid
residues > changes in intramolecular attractions > structure change
Explain the structure/features of chymotrypsin and the significance of
catalytic residues
features:
serine protease family of endopeptidases - shows acid base and covalent
catalysis, inc rate 10^9, 3 chains linked by disulphide
cleaves preferentially next to carboxyl group of aromatic AAs eg. try, try,
phe, sometimes met
catalytic triad:
serine 195 CH2OH pKa 14
histidine 57 CNH pentagon pKa 6
aspartate 102 C=OO- pKa 4 - H bonds to histidine HN holding H in correct
orientation to attract ser proton
Describe the mechanism of chymotrypsin
1. his removes proton from ser
2. O- ser (alkoxide) performs nucleophilic attack on C of peptide (bound non
covalently in hydrophobic pocket) and bonds = tetrahedral transition state
Summary 3
, 3. his proton donated to amine group on c terminal which separates (C-N
cleaved) = RNH2 first product and acyl enzyme intermediate with ester
bond to ser (initial burst phase)
4. H2O binds his > hydrolysis, nucleophilic attack of acyl O= > H to his and
OH to acyl-enzyme = 2nd tetrahedral transition state
5. RCOOH carboxyl group separates (acyl cleaved) forming second product
and regenerating original enzyme (H added back to ser)
How does the rate of an enzyme reaction depends on the concentration of the
substrate(s)?
many show saturation/are hyperbolic ie. at low conc, directly proportional
to rate but at high conc independent
ES complex more stable than E alone so E will show less denaturation/rate
dec at high temps if there is more substrate
Give the MM equation and the three assumptions
V0 = Vmax(S)/(Km + S) = hyperbole
Summary 4
ENZYMES
What is the concept of catalysis? Include 4 catalytic mechanisms
catalysis = increase rate of reaction (both directions) without affecting
final equilibrium of reactants and products or dG eg. catalase 10^14x faster
to a Kcat of 40000000s-1
reduces activation energy of reaction (difference between free energy of
ground and transition state)
1. acid base catalysis - using proton from environment/substrate/active site
to stabilise unstable charged intermediates to lower Ea, make stronger
nucleophiles/electrophiles
2. covalent catalysis - forms transient bond between E and S to make
intermediate with lower activation pathway
3. bond strain - forcing substrate into conformation that strains bonds to inc
likelihood of transition state formation
4. metal ion catalysis - help orientate substrate, stabilise charged
intermediates, mediate redox
Definitions of transition state, isoenzymes, co factors, coenzymes,
intermediate
transition state = unstable, high energy, transient intermediate (highest
energy state is RDS), equally likely to decay to substrate and product
intermediate = compound formed along the pathway with a transient
lifetime eg. ES complex
isoenzymes = catalyse same reaction but different structure/region
(different gene coding and regulation), may have different pH/temp
optimums and Km/Vmax etc but same Keq
cofactors = non protein helper needed for function, inc organic co
enzymes and inorganic ions eg. Cu+ for cytochrome oxidase
Summary 1
, coenzymes = organic, called prosthetic group if tightly bound to enzyme,
transient carriers of atoms/functional groups eg. FAD (from riboflavin)
transfers electrons
What is the meaning of substrate specificity? Give three models to
demonstrate
specificity = enzymes only act on one type of reaction - one substrate or
class of similar molecules, one stereoisomer eg. alcohol dehydrogenase
oxidises primary alcohols to aldehydes
specific residues in active site are important - eg. glyceraldehdye and G3P
both reacted with triose phosphate isomerase but the phosphate group
shows 80% rate acceleration for G3P
1. lock and key (fisher) = shape of substrate is complementary to shape of
enzyme active site
2. induced fit (koshland) = shape of enzyme active site changes upon
binding (induced by weak interactions with substrate), bringing catalytic
residues into position eg. hexokinase binding glucose and MgATP
3. new model = active site is complementary to the transition state - releases
free binding energy when complex forms = stabilises, lowers Ea, reduces
entropy and desolvates substrate
(specific activity = purity of enzyme preparation ie. how many enzyme
units per amount of protein)
What are the 6 classes of enzymes and examples?
classes (HOTLIL)
1. hydrolases eg. trypsin splits Lys-Val - use H2O to split into two molecules
2. oxidoreductases eg. lactate dehydrogenase: pyruvate + NADH + H+ >
lactate + NAD+ (ie. transfer of hydride H- ion) redox transfer of H/O/e
3. transferases eg. alanine aminotransferase (glutamate + pyruvate <> L-
alanine + a-ketoglutarate) or hexokinase - transfer functional groups
between molecules
Summary 2
, 4. lyases eg. aldolase splits F16BP to DHAP and G3P, dopa decarboxylase -
split/join molecules by breaking covalent bonds
5. isomerases eg. phosphoglucose isomerase G6P>F6P - move atoms within
one molecule
6. ligases eg. DNA ligase - join two molecules by forming new covalent
bonds C-C/S/O/N and hydrolysing ATP
further classed by specific type of reaction to give 4 digit number
What is the effect of pH and temp on enzyme activity
pH and temp alter the bonds holding the enzyme in its specific shape,
destroying the active site and catalysis - different enzymes have different
optimums
pH change > (de)protonation of side chains of amines/carboxylic acid
residues > changes in intramolecular attractions > structure change
Explain the structure/features of chymotrypsin and the significance of
catalytic residues
features:
serine protease family of endopeptidases - shows acid base and covalent
catalysis, inc rate 10^9, 3 chains linked by disulphide
cleaves preferentially next to carboxyl group of aromatic AAs eg. try, try,
phe, sometimes met
catalytic triad:
serine 195 CH2OH pKa 14
histidine 57 CNH pentagon pKa 6
aspartate 102 C=OO- pKa 4 - H bonds to histidine HN holding H in correct
orientation to attract ser proton
Describe the mechanism of chymotrypsin
1. his removes proton from ser
2. O- ser (alkoxide) performs nucleophilic attack on C of peptide (bound non
covalently in hydrophobic pocket) and bonds = tetrahedral transition state
Summary 3
, 3. his proton donated to amine group on c terminal which separates (C-N
cleaved) = RNH2 first product and acyl enzyme intermediate with ester
bond to ser (initial burst phase)
4. H2O binds his > hydrolysis, nucleophilic attack of acyl O= > H to his and
OH to acyl-enzyme = 2nd tetrahedral transition state
5. RCOOH carboxyl group separates (acyl cleaved) forming second product
and regenerating original enzyme (H added back to ser)
How does the rate of an enzyme reaction depends on the concentration of the
substrate(s)?
many show saturation/are hyperbolic ie. at low conc, directly proportional
to rate but at high conc independent
ES complex more stable than E alone so E will show less denaturation/rate
dec at high temps if there is more substrate
Give the MM equation and the three assumptions
V0 = Vmax(S)/(Km + S) = hyperbole
Summary 4
