Professional Documents
Culture Documents
Enzymology
E+S ↔ ES → E+P
Learning Objectives for the
Chapter
Diagnostic Enzymology
Introduction (enzymology from a clinical point of view)
Classification and Nomenclature of enzymes
Mechanism of enzymes action
Nature of enzymes regarding energy requirements of
chemical reaction
Enzyme kinetics (substrate concentration, temperature,
cofactors, coenzymes, inhibitors, pH)
Enzyme Assay Techniques
Outline
Allosteric site
A cavity other than the active site
May bind regulator molecules &, thereby, be significant to the
basic enzyme structure
Properties of Enzymes
Are not consumed in reactions they catalyze;
but are rather released at the end of the reactions to be
recycled.
High catalytic efficiency:
Reaction rates 105 – 1017
E.g; peptidases
Catalytic Mechanism of
Enzymes
Before changed into P, the S must overcome an "energy
barrier" - activation energy.
The energy required to raise all molecules in 1 mole of a
compound to the transition state
Answer: Enzyme
2.1 Enzyme Kinetics
Introduction
Definition of Enzyme Kinetics:
The study of the rate of enzyme catalyzed reactions.
i.e., the rate of P formation from S that has bound
specifically to E
15
½ V max
(Reaction follows first-order kinetics)
10
Not all of the active site of
an E are bound by S
Km 10 20
Substrate Concentration = [S]
Direct
If the S or P has a characteristic Abs or spectral
"fingerprint," a coloured or fluorescent
the action of the E itself can be followed by changes in
Abs (e.g. NAD(P)H at 340nm with dehydrogenases),
Enzyme Activity….
Most enzymes do not produce any change in a readily detectable physical
parameter by their activity.
This can be overcome using a coupled assays
versa.
E.g, the enzyme hexokinase, can be assayed by coupling its
production of glucose-6-phosphate to NADPH production,
using glucose-6-phosphate dehydrogenase (G6PDH).
53
Measuring an Analyte Using an
Enzyme
ALT
L-Alanine + 2-oxoglutarate Glutamate + Pyruvate
LDH
NADH + H+ + Pyruvate Lactate + NAD+
Answer: ½ V max
Summary: Enzyme Kinetics –
Continued
Michaelis-Menten curve describes constant Km,
the substrate conc. that corresponds to ½ V max
– the maximum reaction velocity or rate.
Lineweaver-Burk transformation gives inverse:
1/Vmax and 1/Km
But yields a linear line instead of a hyperbolic
curve.
Question for You:
Answer: Competitive
Summary: Enzyme Kinetics –
Continued
The reaction rate increases with substrate
concentration in first-order kinetics. The rate
stabilizes when there is an excess of substrate –
zero-order kinetics.
Some enzymes require cofactors or activators,
often metallic ions, smaller proteins or vitamins.
Question for You: