Objectives
List
General Characteristics
Most enzymes are globular protein catalysts that increase the
velocity of a chemical reaction, and are not consumed during
the reaction they catalyze
Non protein enzymes exists RNA molecules (ribozymes)
Linear chains of amino acids folded into 3-D macromolecules
Unique amino acid sequence produces specific enzyme with
unique properties for binding to substrate.
A)Active sites; A special pocket containing amino acid side
chains that create a three-dimensional surface complementary
to the substrate
Classification of enzymes
A)Recomended name: Many enzymes have been named by
adding the suffix -ase to the name of their substrate
(Glucosidase) or to a word or phrase describing their activity
(lactate dehydrogenase).
Some enzymes retain their original trivial (trypsin and
pepsin)
A)Systematic name; International Union of Biochemist (IUB)
system divides enzymes into six functional classes, each with
subclasses, based on the type of reaction catalyzed
No.
Class
1)
Oxidoreductases
2)
Transferases
3)
Hydrolases
4)
Lyases
5)
Isomerases
6)
Ligases
Substrate is converted in a
product through an
intermediate state
(Transition state). Energy
required to reach the
transition state defines the
rate of reaction.
Enzyme Kinetics
Enzymes accelerate reactions by lowering the free energy
of activation
Enzymes do this by binding the transition state of the
reaction better than the substrate
Is the investigation of how enzymes bind substrates and
turn them into products.
Reactants and products conc. are usually 100 to 1000 times
greater than enzyme conc.
Enzyme-substrate complex (ES);
Transition state (ES*);
Enzyme-Product complex (EP)
Product (P) and free enzyme (E)
E+S
ES
ES*
EP
P+E
Temperature
Increase of velocity with temperature
Decrease of velocity with higher temperature
pH
The ionization of the active site
enzyme denaturation
k1
E+S
ES
k-1
k2
E+P
(k-2 is insignificant early in rxn)
Vo = k2 [ES]
[ES] =
[Etotal][S]
________________________
[Etotal][S]
____________
KM + [S]
k1
Vo = k2 [ES]
Vo =
k2 [Etotal][S]
____________
KM + [S]
Vo = Vmax when [Etotal] = [ES]
(at saturation)
Vo =
____________
KM + [S]
Vmax[S]
_________
Km + [S]
When [S] is low, the equation for rate is first order in [S]
Vo =
Vmax[S]
____________
KM + [S]
KM = [S]
when Vo =
Vmax
_____
2
From Lehninger
Principles of Biochemistry
Km is the substrate
concentration that
corresponds to Vmax
2
V0
Substrate added
(mmol/min)
(mmol/L)
216
0.9
323
2
435
4
489
6
647
2,000
Without graphing
Vmax = 647
Vmax /2 = 647 / 2 = 323.5
Km = 2 mmol/L
Understanding Km
Km is a constant
Km is a constant derived from rate constants
Km is, under true Michaelis-Menten conditions,
an estimate of the dissociation constant of E
from S
Small Km means tight binding; high Km means
weak binding
Enzyme
Substrate
Km (mM)
Glutamate dehydrogenase
NH4+
Glutamate
CO2
57
0.12
12
Carbonic anhydrase
Understanding Vmax
The theoretical maximal velocity
Vmax is a constant
Vmax is the theoretical maximal rate of the reaction - but
it is NEVER achieved in reality
To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate
Vmax is asymptotically approached as substrate is
increased
1
______
Vo
KM
+ ______
Vmax[S]
Vmax
_______
From Lehninger
Principles of Biochemistry
Values of kcat range from less than 1/sec to many millions per sec
1.1
Mutant R160K
0.015
Enzyme Inhibitors
Reversible versus Irreversible
Reversible inhibitors interact with an enzyme via
noncovalent associations
Irreversible inhibitors interact with an enzyme via covalent
associations
Classes of Inhibition
Two real, one hypothetical
Competitive inhibition - inhibitor (I) binds only to E, not to ES
Uncompetitive inhibition - inhibitor (I) binds only to ES, not to
E. This is a hypothetical case that has never been
documented for a real enzyme, but which makes a useful
contrast to competitive inhibition
Noncompetitive (mixed) inhibition - inhibitor (I) binds to E and
to ES
Enzyme Inhibition
From Lehninger
Principles of Biochemistry
Competitive
Inhibition
Kmchanges
while Vmax
does not
Uncompetitive
Inhibition
Km and Vmax
both change
Noncompetitive
(Mixed) Inhibition
Km and Vmax
both change
From Lehninger
Principles of Biochemistry
Malonateisastrong
competitiveinhibitorof
succinatedehydrogenase
From Lehninger
Principles of Biochemistry
Competitive Inhibition
Kmchanges while Vmax
does not
+I
1/V
No I
-1 / Km
-1 / Kmapp
1/[S]
Where Kmapp = Km
= 1 + [I]
KI
Uncompetitive inhibition
1/V
/Vmax
+I
No I
- / Km
/Vmax
1/[S]
-1 / Km
= 1 + [I]
KI
Vmaxapp
KMapp
No inhibitor
Vmax
KM
Competitive
Vmax
KM
Uncompetitive
Vmax/
KM/
Noncompetitive (Mixed)
Vmax/
KM/
= 1 + [I]
= 1 + [I]
KI
K I
Regulatory Enzymes
important in controlling flux through metabolic pathways
1. Allosteric enzymes
From Lehninger
Principles of Biochemistry
Enzymes in diagnosis
Generally enzymes are present in cells in a higher conc than body fluids
Enzymes in serum is an indication of tissue or cellular damage
Commonly assayed enzymes are the amino transferases
Alanine aminotransaminase (ALT)
Aspartate aminotransferase (AST)
Lactate dehydrogenase (LDH)
Creatinine kinase (CK)
Gammaglutamyl transpeptidase (GGT)
Home work
Enzyme
Alkaline Phosphatase
Acid Phosphatase
Gammaglutamyl
transpeptidase (GGT)
Alanine
aminotransaminase
(ALT)
Aspartate
aminotransferase
(AST)
Lactate
dehydrogenase (LDH)
Creatinine kinase (CK)
Principal tissue
Normal level
Public health