Prof Dr Koh Chong Lek

Supplementary lecture notes (September 2016)

Bioch L10
Properties & Kinetics of Enzymes
Understand the following terms and concepts that you had learnt
Reactant
Product
Energy barrier
Activation energy, Ea
Free energy, G
Catalyst
Chemical catalyst
Biocatalyst
Metabolism
Anabolism
Catabolism
Enzyme
IUBMB classification of enzymes
IUBMB classification number
Ribozyme
Zymogen or proenzyme
Proteolytic cleavage or proteolysis
Cofactor
Activator
Coenzyme
Prosthetic group
Holoenzyme
Apoenzyme
Cytosol
Organelle
Primary (1) structure of a polypeptide
chain/protein
Secondary (2) structure of a polypeptide
chain/protein
Tertiary (3) structure of a polypeptide
chain/protein
Quaternary (4) structure of a protein
helix (helices)
sheet (sheets)
Monomer, monomeric
Multimer, multimeric
Homomultimeric (e.g., or 2)
Heteromultimeric (e.g., )
Enzyme specificity
Enantiomers, optical isomers, or
stereoisiomers
Active site

Enzyme-substrate (ES) interaction

ES complex
Enzyme-product (EP) complex
Lock-and-key model
Induced-fit model
Enzyme activity
International unit (IU)
Enzyme kinetics
Reaction rate (velocity)
First order reaction
Zero order reaction
Michaelis-Menten equation, Km (the Michaelis
constant)
Significance of Km
Km versus affinity for substrate
Hyperbolic curve
Sigmoid curve
Lineweaver-Burk plot or Double reciprocal
plot
Gradient or slope
x-intercept
y-intercept
Optimum temperature
Optimum pH
Optimum ionic strength
Denaturation of enzyme
Inhibition, inhibitor
Reversible inhibition
Irreversible inhibition
Competitive inhibition
Noncompetitive inhibition
Uncompetitive inhibition
Structure analogue
Structural resemblance
Inhibitor site
Noncovalent binding
Covalent binding
Isozyme or isoenzyme
Isoform
Metabolic pathway
Precursor
Intermediary metabolite/product

Regulatory enzyme
Allosteric protein/enzyme
Active/catalytic site
Allosteric/regulatory site
Allosteric effector/modulator
Allosteric interaction
Allosteric transformation
Negative effector
Positive effector

End product feedback inhibition or

End product inhibition
Threonine
Threonine deaminase
-ketobutyrate
Isoleucine
Phosphorylation, dephosphorylation
Adenylylation
Methylation
Cascade of zymogen activation

Bioch L11
Clinical Enzymology
Enzymes
Biological catalysts or biocatalysts
Very efficient can increase reaction rates at the order of x10
Almost all are proteins so liable to denaturation
Specific to substrates
Partly specific to tissues
Assay by measuring the rate of specific reaction catalysed by that enzyme
Enzymes are used as diagnostic indicators we routinely determine selected plasma enzyme
activities in order to diagnose diseases of the heart, liver, skeletal muscle, pancreas, and other
tissues
Enzymes are used as therapeutic agents we use enzymes as drugs, enzyme therapy
Enzymes are used as diagnostic/analytical tools we use enzymes as chemicals in clinical laboratory
assays
Enzymes in clinical diagnosis
Enzymes
Secretory - produced by tissues (eg, liver), acting in plasma
Biomarkers
A biomarker is a clinical laboratory test which is useful in detecting dysfunction of an organ or
detecting a particular disease state
Different specific biomarkers are used to detect their respective diseases
Liver diseases
Enzymes commonly used for diagnosis of liver diseases are alanine transaminase (ALT; also known as
alanine aminotransferase) and aspartate transaminase (AST; also known as aspartate
aminotransferase), especially the former which increases markedly in liver diseases
Other enzymes used: alkaline phosphatase (ALP) and glutamyltransferase ( GT)

Myocardial infarction
Myocardial infarction (MI) (i.e., heart attack) is the death (necrosis) of heart muscle/myocardial cells
secondary to prolonged lack of oxygen supply (about 30 minutes of anoxia)
Three key enzymes in blood plasma of MI patients show elevated levels
They are creatine kinase (a dimer, CK-MB), aspartate aminotransferase (a homodimer, AST), and
lactate dehydrogenase (LDH1 a tetrameric protein, H4, HHHH)
Among them, CK-MB and AST show marked increase in activity within the first two days of acute MI
Whereas LDH1 shows marked increase in activity much later

The main factor that accounts for the early and late elevations in enzyme activities in serum
following MI is the rate of diffusion of each enzyme through damaged tissue into the circulation
Diffusion rate is determined by molecular size and is related to molecular weight

The molecular weights of CK, AST, and LDH are 80, 90, and 140 kilodaltons (kDa), respectively
It is evident that CK diffuses out of the necrotic region earliest and most rapidly and LDH latest and
most slowly
Currently, cardiac biomarkers routinely measured in the clinical laboratory for the purpose of
diagnosing and monitoring MI are cardiac troponin I, cardiac troponin T (both of which are not
enzymes), and CK-MB
AST and LDH were previously used as cardiac biomarkers of MI, but are no longer used in clinical
practice

Understand the following terms and concepts that you had learnt
Secretory enzyme
Intracellular enzyme
Tissue specific enzyme
Physiological enzyme level
Alteration of enzyme plasma level
Diagnostic enzymology
Enzymes assayed for diagnostic purposes
Isoenzymes assayed for diagnostic purposes
Isoenzymes
Enzyme activity, international unit (IU)
Coupled assay
Differences in ultraviolet absorption spectra of NADH/NADPH and NAD+/NADP+
Liver diseases
Myocardial infarction - definition
Myocardial infarction key enzymes elevated and the main factor that accounts for the early and
late elevations in enzyme activities in serum following MI
Lactate dehydrogenase (LDH) and its isoenzymes
Creatine kinase (CK) and its isoenzymes
Alanine transaminase (ALT; also known as alanine aminotransferase)
Aspartate transaminase (AST; also known as aspartate aminotransferase)
Alkaline phosphatase (ALP)
Acid phosphatase (ACP)
Amylase
glutamyltransferase ( GT)
Enzymes as tumour markers
Inborn errors of metabolism
Congenital/Inherited metabolic diseases
Therapeutic uses of enzymes
Analytical/Laboratory uses of enzymes

Bioch L12
Carbohydrate Structure & Function
Understand the following terms and concepts that you had learnt
Polyhydroxy aldehyde (aldose)

Polyhydroxy ketone (ketose)

Carbonyl group
Aldehyde (CHO)
Ketone (C=O)
Monosaccharide
Glycosidic bond
Disaccharide
Oligosaccharide
Polysaccharide
Glycan
Homopolysaccharide, homoglycan
Heteropolysaccharide, heteroglycan
Triose, glyceraldehyde, dihydroxyacetone
Tetrose, erythrose, erythrulose
Pentose, arabinose, ribose, xylose, ribulose, xylulose
Hexose, glucose, galactose, mannose, fructose,
Heptose, glucoheptose, sedoheptulose
Disaccharide, maltose, lactose, sucrose
Trisaccharide, raffinose
Tetrasaccharide, stachyose
Pentasaccharide, verbascose
Starch, dextrin, glycogen, cellulose, dextran, chitin
Agar, gum, pectin, glycosaminoglycan or mucopolysaccharide
Straight chain structure
Ring or cyclic form (Fischer structure, Haworth structure)
Boat and chair forms
Anomeric carbon
Epimeric carbon
Penultimate carbon
Terminal carbon
D-glucose, -D-glucopyranose (Fischer structure), -D-glucopyranose (Haworth structure)
[SORRY, slide 18 has three typographical errors: should be glucopyranose, Fischer, and Haworth]
Structural isomerism
Isomers
Aldose-ketose isomerism
Stereoisomerism, stereoisomers
Asymmetric carbon atoms
D and L isomerism, enantiomers
Optical isomerism, optical isomers
Racemic or dl mixture
Epimerism, epimers
Epimers of D-glucose: D-galactose and D-mannose
Anomerism, and anomers
Mutarotation
Furfural derivative
Enediol
Enolisation
Sugar acids
Aldonic acid
Gluconic acid
Saccharic or alderic acid

D-glucosaccharic acid (D-glucaric acid)

D-mannosaccharic acid (D-mannaric acid)
D-galactosaccharic acid (D-galactaric acid)
Uronic acid
Glucuronic acid
Sugar alcohol
Polyhydroxy alcohol
D-sorbitol, D-mannitol, D-dulcitol (D-galactitol), D-ribitol, D-xylitol
Osazone
Glucosazone, lactosazone, maltosazone
Glycoside
O-glycosidic (glycosyl) and N-glycosidic (glycosyl) bonds or linkages
Derivatives of monosaccharides
Reducing disaccharides: maltose, lactose, isomaltose, cellobiose
Non-reducing disaccharides: sucrose, trehalose
Maltose
(14) glycosidic bond (a form of O-glycosidic bond)
Maltase
Lactose
(14) glycosidic bond (a form of O-glycosidic bond)
Lactase
-D-galactosidase
Sucrose
(12) glycosidic bond (a form of O-glycosidic bond)
Sucrase
Isomaltose
(16) glycosidic bond (a form of O-glycosidic bond)
Isomaltase
Trehalose
(11) glycosidic bond (a form of O-glycosidic bond)
Trehalase
Starch, homopolysaccharide or homoglycan
Amylose, linear homopolysaccharide or homoglycan, (14) glycosidic bonds
Amylopectin, branched homopolysaccharide or homoglycan, (14) and (16) glycosidic bond
bonds
Amylase
Dextrins: amylodextrins, erythrodextrins, and achrodextrins
Glycogen, highly branched homopolysaccharide or homoglycan, (14) and (16) glycosidic bond
bonds
Degradation of glycogen (glycogenolysis) glycogen phosphorylase, (14) to (14)-glucan
transferase, amylo-(16)-glucosidase [See Slide 22, Bioch L13 Carbohydrate Metabolism]
Cellulose, linear homopolysaccharide or homoglycan, (14) glycosidic bonds
Cellulase
Inulin, (1-2) glycosidic linkages
Inulinase
Mucopolysaccharide, heteropolysaccharide or heteroglycan
Glycosaminoglycan
Proteoglycan
Hyaluronic acid, chondroitin sulphate, keratin sulphate, heparin, heparan sulphate
Glycoprotein
Transferrin, ceruloplasmin

Bioch L13
Carbohydrate Metabolism
Slide 5:

Slide 7:

Understand the following terms and concepts that you had learnt
Mastication
Ptylin
Endoglycosidase
Mouth: dextrins, maltotriose, maltose, isomaltose
Small intestine, pancreatic -amylase: -limit dextrins (3 to 5 glucose units, including maltotriose),
maltose, and isomaltose
Small intestine, enzymes located in the cells of brush border: glucose, fructose, galactose
Absorption of monosaccharides

Metabolic fate of monosaccharides

Glycolysis
Embden Meyerhof pathway
Aerobic glycolysis, 1 mole of glucose (6C) is split into 2 moles of pyruvate (3C), which is later
oxidatively decarboxylated by pyruvate dehydrogenase to acetyl CoA (2C) citric acid cycle
Anaerobic glycolysis, 1 mole of glucose (6C) is split into 2 moles of pyruvate (3C), then to 2 moles of
lactate (3C) Cori cycle or lactic acid cycle in the liver
Glycolysis I
Glycolysis II
ATP generated by substrate level phosphorylation
ATP generated by the oxidative phosphorylation of NADH
Oxidation of 1 mole of NADH produces 3 moles of ATP
Aerobic glycolysis: Glucose + 2 Pi + 2 ADP +2 NAD+ 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O
Anaerobic glycolysis: Glucose + 2 Pi + 2 ADP 2 Lactate + 2 ATP + 2 H2O
Tricarboxylic acid cycle, citric acid cycle, or Krebs cycle
Pyruvate (3C) acetyl CoA (2C)
Acetyl CoA (2C) + oxaloacetate (4C) [a component of TCA] citric acid or citrate (6C) [a component
of TCA] TCA
Cori cycle or lactic acid cycle: Lactate produced by anaerobic glycolysis in the muscles in the liver is
converted to glucose (gluconeogenesis)
Pentose phosphate pathway/shunt or hexose monophosphate (HMP) pathway/shunt
Oxidative and non-oxidative phases
Slide 17:

Gluconeogenesis, from pyruvate to glucose

Glycogenesis, from glucose to glycogen
Glycogenolysis, from glycogen to glucose
Slide 22:
Correction: change (4)(14)-glucan transferase to (14) to (14)-glucan transferase
Uronic acid pathway or glucuronic acid cycle
Galactose metabolism

Metabolism of alcohol
When alcohol is consumed, most of it is absorbed by intestine and transported to the liver where it
is oxidised by alcohol dehydrogenase, an NAD+ dependent enzyme, to acetaldehyde.
Another NAD+ dependent enzyme, acetaldehyde dehydrogenase, oxidises acetaldehyde further to
acetate, which is subsequently converted to acetyl CoA.

The activity of alcohol dehydrogenase is more than that of acetaldehyde dehydrogenase. Hence,
acetaldehyde accumulates in liver. Acetaldehyde is toxic, which in excess may lead to cell death.
Excess acetaldehyde contributes to hangover symptoms.
Many Orientals/Asians (e.g., Chinese, Japanese, Koreans) have less functional or lack an active
acetaldehyde dehydrogenase and are unable to metabolise acetaldehyde, which then accumulates
in their liver. As a result, after their first drink of alcohol, they become flushed (i.e., their face turns
red) and they get sick to their stomach easier than Westerners who have this enzyme.
Both the oxidation steps of alcohol produce NADH, resulting in high NADH/NAD+ ratio, which can
cause many health problems. NADH is an electron transporter. Its presence causes liver cells to
decrease creation of glucose (gluconeogenesis), to increase breakdown of glucose (glycolysis), to
decrease fat breakdown and increase fat synthesis (resulting in fatty liver and steatosis). NADH is
also used to convert pyruvate to lactate, which means high levels of NADH lead to increased
anaerobic glycolysis and lactic acidosis.

http://www.stomponstep1.com/alcohol-metabolism-methanol-poisoning-fatty-change/
The Effects of Alcohol in the Body (INFOGRAPHIC)
http://www.huffingtonpost.com/2012/12/28/alcohol-effects-body-infographic_n_2333328.html

Bioch L14
Carbohydrate Regulation
Understand the following terms and concepts that you had learnt
Hormonal control of digestion
Hormone, location, function
Regulation of glycolysis
Regulation of citric acid cycle
Regulation of pentose phosphate pathway (PPP)
Insulin enhances PPP by inducing G-6-PD & 6-phosphogluconate dehydrogenase (Slide 13)
Regulation of gluconeogenesis
Regulation of glycogenesis and glycogenolysis

Bioch L15
Electron Transport/Oxidative Phosphorylation I
Understand the following terms and concepts that you had learnt
Exergonic reaction
Endergonic reaction
Cellular or aerobic respiration: ATP synthesis and glucose metabolism
C6H12O6 + 6 O2 + 36 Pi +36 ADP + 36 H+ 6 CO2 + 36 ATP + 42 H2O
Redox reactions: oxidation & reduction
e- donor, reducing agent
vs
e- acceptor, oxidising agent
+
Reduction of NAD to create NADH
Flavin structure and nomenclature: FMN, FMNH, FMNH2, FAD, FADH, FADH2
ATP: High energy compound
The ATP cycle, coupled reactions
An overview of cellular respiration
Substrate-level phosphorylation
Oxidative phosphorylation (oxphos)
Glycolysis
Glycolysis I
Glycolysis II
Oxidation of pyruvate to acetyl CoA
Citric acid cycle
Stages of cellular respiration: Three stages of harvesting of energy from glucose
Oxidation of glucose during aerobic respiration: 1 glucose 10 NADH, 2 FADH2, 6 CO2, 4 ATP
Oxidative phosphorylation (oxphos): ATP synthase, proton gradient
Chemiosmosis
Mitochondria
Mitochondrial electron transport chain (ETC)
Protein complexes (designated I to IV) with prosthetic groups
Cytochromes b, c1, c, and aa3
The sequence of electron carriers in ETC

Free energy change during electron transport

Oxidative phosphorylation: Oxidation step (ETC) + Phosphorylation step (chemiosmosis)
Summary: Glucose to CO2, H2O, ATP
Oxidation of glucose during aerobic respiration

Bioch L16
Electron Transport/Oxidative Phosphorylation II
Understand the following terms and concepts that you had learnt
Oxidative phosphorylation: Oxidation step (ETC) + Phosphorylation step (chemiosmosis)
Substrate level phosphorylation: Direct transfer and donation of a phosphoryl (PO32-) group to ADP
or GDP from a phosphorylated reactive intermediate
Inhibitors of ETC
Inhibitors of oxidative phosphorylation
Uncouplers of oxidative phosphorylation
Shuttle systems for oxidation of extramitochondrial NADH
The glycerol-phosphate shuttle
The malate-aspartate shuttle
Inherited defects of oxphos

Bioch L21 & L22

Membrane Biochemistry I & II
There are many terms and concepts that you had learnt
Please go through my notes + relevant references