Download Enzymes Recap

Enzymes Recap E + S Exergonic (release) Endergonic (absorb) Higher rates Milder condi5ons Greater Specificity Control Capacity ES EP E + P Substrate Accommoda5on ac5ve site environment stabilises/promotes forma=on of the transi=on state intermediate S Lock and Key E Vs S E “Induced Fit” Dynamic recogni5on Enzyme-­‐substrate hydrogen bonding assists In free energy release and underpins the reduc=on in ac=va=on energy observed in the presence of enzyme Mechanisms of Catalysis at Ac5ve Site Apoenzyme + cofactor  Holoenzyme Metals/ions coenzymes Types of Enzymes & Nomenclature Enzyme kine5cs Rates of Enzyma5c Reac5on Temperature op=mum in humans is circa 37˚C pH op=mum generally between 5 and 9 (N.B. pepsin is auto-­‐ac=vated from a precursor in the stomach in HCl condi=oned low pH) N.B. Think of effect of temperature and pH on protein folding and ionisa=on of amino acid side chains AND HENCE ACTIVE SITE STRUCTURAL CHEMISTRY (For Monomeric Enzymes) Reac=on rate increases upon increasing substrate un=l satura=ng concentra=ons of substrate Increasing enzyme concentra=on in presence of excess substrate elevates the point at which the maximum rate of reac=on is reached Useful Kine5c Data (Monomeric Enzymes) Michaelis and Menton (1913) Vmax-­‐Rate of reac=on (product forma=on/unit =me) when enzyme is saturated High Vmax=rapid catalysis Km-­‐ Substrate concentra=on yielding 1/2 Vmax. Describes the affinity of an enzyme for substrate. Low Km=high affinity Lineweaver-­‐Burk Plots Plot reciprocals 1. Reversible Enzyme Inhibi5on 1)  Compe==ve e.g) ibuprofen 2) Uncompe==ve e.g. herbicides 3) Non-­‐compe==ve e.g. deoxycyline 2. Irreversible Enzyme Inhibi5on -­‐Group-­‐specific (react with specific ac=ve site amino acid side chains) -­‐Reac/ve substrate analogues (covalent binding at ac=ve site) -­‐Suicide inhibitors (substrate or transi=on state analogues genera=ng a reac=ve intermediate that inac=vates enzyme via covalent modifica=on) Physiological Regula5on of Enzyme Ac5vity 1. Allosteric Control -­‐Product feedback effect on rate limi=ng step in pathway 2. Isoenzymes Same reac=on, more than one enzyme with differing characteris=cs e.g. Km 3. Reversible Covalent modifica5on Phosphoryla=on 4. Proteoly5c ac5va5on Gastrointes=nal zymogens 5. Enzyme synthesis and degrada5on Transcrip=on, tagging and proteosomal degrada=on Your Learning From Today Should focus on being able to; 1)  Describe how enzymes affect the rate of biochemical reac=ons and why this is useful in the body 2)  Classify enzyme types and mechanisms of enzyma=c catalysis 3)  Outline how enzyme ac=vity can be quan=ta=vely measured through plots of substrate concentra=on versus product forma=on (enzyme kine=cs) 4)  Explain the mechanisms relevant in the pharmacological and physiological regula=on of enzyme ac=vity Cellular Respiration
Glycolysis
Kreb’s Cycle
ETC
PG1005
Lecture 11
Glycolysis
My Teaching Objec5ves •  To discuss why when the topic of cellular energy produc=on is introduced, we use glucose as the fuel of choice for the genera5on of ATP. •  To revise the general mechanisms of glucose uptake. •  To describe the enzyma5c reac5ons occurring at each step of glycolysis. (substrates, enzymes, energy transforma=ons, reac=on types, products) •  To highlight the existence of checkpoints in glycolysis which permit physiological supervision of flux through the process Why is ATP The Cellular Energy Source? •  Many cell processes are not energe=cally favourable and are thus require energy input to drive endergonic reac=on The heat energy (free enthalpy)
released from ATP can be
harnessed to give the activation
energy required to drive reactions
Hydrolysis of bonds
This energy used to power
cellular processes
ATP can be MADE from
ADP + Pi
requires energy input
ATP – ADP cycle
Synthesis
Glucose-­‐Origin as a Fuel Source Dietary Complex Carbohydrate (starch,glycogen) Sucrose
Lactose
Salivary and
pancreatic amylase
sucrase
lactase
Disaccharide Trisaccharide Limit dextrin
(maltose)
(maltostriose)
maltase
GLUCOSE
α-glucosidase
α-dextrinase
GLUCOSE
GLUCOSE
GLUCOSE
GALACTOSE
FRUCTOSE
Why Consider Glucose U5lisa5on? Glucose vs Monosaccarides
glucose
•  Product of prebio=c chemistry? •  Due to persistence in ring structure rela=ve to monosaccarides, does not modify protein structure (glyca=on) carbonyl-­‐amino group reac=ons "" " " " Schiff base  amino-­‐ketone •  The chemical structure is such that its successive oxida5on yields high energy electrons that can be harnessed to drive ATP synthesis in an energy efficient manner Generic Glucose Uptake •  Dis=nct from energy dependent sodium glucose transporters which work against gradient (e.g small intes=ne) •  5 classes (GLUT1-­‐5) •  12 transmembrane helix structure •  GLUT 1 and 3 are principal basal entry pathways •  Operate by “flipping” glucose across membrane •  As glucose is consumed intracellularly, favourable gradient means energy independence Glycolysis-­‐Defini5on •  A sequence of 10 reac=ons based on using glucose as a substrate to generate two molecules of pyruvate. •  Anaerobic, common in all cells •  For each molecule of glucose a net produc=on of 2 ATP occurs. Lactic acid fermentation
Oxidative phosphorylation
Gluconeogenesis
Adapted from Figure 2-10 Human Physiology Cells to Systems (7th Ed.) Sherwood L. p245
Glycolysis – 2 stage process •  Stage 1 -­‐ no ATP genera=on -­‐ Glucose  Fructose 1, 6-­‐bisphosphate -­‐ in 3 steps -­‐ phosphoryla=on -­‐ isomeriza=on -­‐ 2nd phosphoryla=on -­‐ Fructose 1, 6-­‐bisphosphate  3C interconvertable units Main aim: trap glucose in cells & form a compound
that can be cleaved into phosphorylated 3C units
Glycolysis -­‐ Stage 1 ENZYMES Glucose enters cell……….
1.1 Hexokinase
-  phosphoryl transfer
-  traps glucose, why?
-  eg induced fit
(Necessary for generation of
3C downstream intermediates)
1.2 Phosphoglucose Isomerase
-  isomerisation
-  ring structure broken
-  ring reformed (position of atoms changed)
6 to 5
membered
ring
1.3 Phosphofructokinase
-  phosphoryl transfer (bis, 2 P)
-  allosteric enzyme (sets pace)
Modified from http://img.sparknotes.com/figures/
Comple5on of Stage 1 Genera=on of 3-­‐carbon GAP intermediate by cleavage of 6C F-­‐1, 6-­‐BP 1.4 Aldolase
-  aldol cleavage
-  reversible
1.5 Triose phosphate isomerase
- Isomerisation
-  max ATP
Entry of
Fructose
as F-1-P
Economy of metabolism
GAP metabolism
drives left to
right reaction
Glycolysis – 2 stage harvest -­‐ so far we’ve spent 2ATP & gained 2GAP -­‐ This is PACKBACK =me -­‐ Series of steps to harvest energy -­‐ lying within GAP -­‐ Harvest ATP Main aim: Harvest energy
Glycolysis: Stage 2.1
Aim: Genera=on of energy rich 3 carbon phosphate donor used to drive substrate level genera=on of ATP 2.1 Glyceraldehyde
Phosphate dehydrogenase
1. oxidation
(GAPDH)
- Sum of 2 processes
1. Aldehyde oxidation
2. phosphorylation
-  favourable/unfavourable processes
-  must be coupled
-  high P transfer potential of products
2. phosphorylation
+H2O
reduction
Nicotinamide adenine
dinucleotide
(B vitamin niacin derivative)
Glycolysis: Stage 2.2
Aim: Substrate level genera=on of ATP Energy rich
Molecule
With high P
transfer
potential
2.2 Phosphoglycerate kinase
(PGK)
- phosphoryl transfer
N.B.
x2 per molecule
of glucose
Due to the action of
??
Glycolysis: Stage 2.3
Aim: Generation of Pyruvate For Oxidative Phosphorylation
Generation of additional ATP
2.3 Phosphoglycerate
mutase
- phosphoryl shift
2.4 Enolase
- dehydration
-  P transfer potential
2.5 Pyruvate kinase
- phosphoryl transfer
Strong phosphate
donor
$
Stable
x2
Net Glycoly5c Reac5on Glucose + 2Pi + 2ADP + 2NAD+ 2 Pyruvate + 2ATP + 2NADH + 2H+ + 2H2O Check Points in Glycolysis In metabolism, enzymes catalysing irreversible reac=ons can become control points 1) PFK -­‐ allosterically inhibited by high ATP pacemaker 2) Backward inhibi=on of hexokinase (nega=ve feedback via glucose-­‐6-­‐phosphate) 3) Pyruvate kinase -­‐ Allosterically inhibited by ATP -­‐ Ac=vated by F1,6BP http://faculty.ksu.edu.sa/70917/Pictures%20Library/glycolysis%20pathway.gif
Gluconeogenesis During fasting and in certain cell types (liver,kidney)
Process aimed at normalising plasma glucose
Essentially synthesis of glucose from non-carbohydrate precursor
GLUCOSE
Glycerol kinase
DIHYDROXYACETONE
Glycerol phosphate
dehydrogenase
Combination of
reversible steps of
glycolysis and new
OXALOACETATE
bypass steps
Protein-Amino acid
TAG-Glycerol
Lactate
PYRUVATE
dehydrogenase
Anaerobic muscle-Lactate
Your Learning From Today Should focus on being able to;
•  Explain why glucose acts as a fuel of choice for the genera=on of ATP. •  Detail how dietary carbohydrate is digested and the general mechanisms of glucose uptake in the gut and beyond. •  List and explain the sequence of enzyma=c reac=ons occurring at each step of glycolysis. (substrates, enzymes, products, reac=on types) •  Demonstrate an understanding of the principle of allosteric control of enzyme ac=vity in glycolysis Kreb’s Citric Acid Cycle
My Teaching Objec5ves •  To discuss the transition from pyruvate generation in the
cytosol to the establishment of electron harvesting reactions
in the mitochondrial matrix
•  To describe the enzymatic reactions occurring at each step
of Kreb’s Citric Acid Cycle (KCAC).
(substrates, enzymes, products, reaction types)
•  To highlight the existence of checkpoints in the KCAC which
permit physiological supervision of flux through the process
Glycolysis to KCAC -­‐ How we got here & des5na5on We
are
here
Adapted from Figure 2-10 Human Physiology Cells to Systems
(7th Ed.) Sherwood L. p245
Glucose  pyruvate
Oxidative
phosphorylation
2 ATP
? More energy?
But first we have to:
-  move to mitochondria
-  transform pyruvate into acetate…….
Pyruvate Symporter
Cytosol
Mitochondrial matrix
New Enzymes
Pyruvate dehydrogenase Complex Aim: Generation of 2 carbon intermediate for subsequent
Generation of citrate (6C)
3 enzyme complex, 5 co-factors
dihydrolipoyl
dehydrogenase
(E3)
1. DECARBOXYLATION
Hydroxyethyl TPP
= cofactor
2. OX
Pyruvate
dehydrogenase
(E1)
IDATIO
N
Lipoamide
pyruvate
By-Products
CO2
NADH
TPP, Thiamine pyrophosphate
=enzyme
3. OXIDATION
CAPS=reaction type
dihydrolipoyl
transferase
(E2)
Dihydrolipoamide
Acetyl CoA acetyllipoamide
CoA
N.B. Do not try to learn the steps on this slide “off by heart”
Cycle Basics •  Harvest high energy electrons and transfer them to electron carriers for use in oxida=ve phosphoryla=on •  Involves the genera=on of a 6 carbon tricarboxylic acid (citric acid) from oxaloacetate (C4) and acetyl CoA •  Sequen=al oxida=on of two carbon units generates CO2, reduced electron intermediates, 1 molecule of guanosine triphosphate (GTP) and regenerates oxaloacetate •  High energy phosphate group transfer from GTP to ADP generates ATP (x2 per molecule of glucose) Kreb’s Cycle Definition as a cycle
is due to regeneration
of oxaloacetate
Adapted from Fig. 2.12 Human Physiology Cells to
Systems (7th Ed.) Sherwood L.
Citrate Synthase Aim: Generation of 6 carbon intermediate for subsequent
coupling of electron transfer to decarboxylation
1) 
2) 
Oxaloacetate condenses with acetyl CoA to form citryl CoA Hydrolysis of Citryl CoA yields citrate and Co A CONDENSATION
+
Binding order
Oxaloacetate-Acetyl-CoA
Enclosure of
active site
HYDROLYSIS
Release order
CoA-Citrate
Citrate Isomerase (aconitase) Aim: Rearrangement of hydroxyl group to facilitate subsequent decarboxyla=on reac=ons Dehydration
Hydration
Isocitrate Dehydrogenase Aim: Genera=on of ALPHA KETOGLUTARATE (5C) and NADH Unstable intermediate
OXIDATION-REDUCTION
DECARBOXYLATION
Alpha ketoglutarate dehydrogenase Aim: Generation of succinyl CoA (4C) and NADH
•  Similar to pyruvate dehydrogenase
(i.e. 3 enzymes, co-factors, addition of CoA, loss of 1C and gain of NADH)
high energy
thioester bond
Succinyl CoA synthetase Aim: Transfer of energy stored in Succinyl CoA to drive GTP formation and
generation of succinate (4C)
•  Succinyl CoA is phosphorylated while in alpha subunit of enzyme releasing CoA •  Subsequently, the alached phosphoryl group is transferred to GDP in beta subunit of enzyme SUBSTRATE LEVEL PHOSPHORYLATION
Succinate to Oxaloacetate Aim -­‐ Oxaloacetate synthesis -­‐ Further synthesis of reduced intermediates OXIDATION
Succinate
dehydrogenase
HYDRATION
fumarase
OXIDATION
malate
dehydrogenase
Net Reac5on of Cycle (Per Acetyl CoA) Acetyl CoA + 3NAD+ + FAD + GDP + Pi + 2H2O 2CO2 + 3NADH + FADH2 + GTP + 2H+ + CoA NUCLEOSIDE DIPHOSPHATE KINASE
GTP + ADP  GDP + ATP
Control Points in Cycle Pyruvate Dehydrogenase •  i) inhibited by high acetyl CoA, ATP and NADH ii) Phosphoryla=ve (kinase) inac=va=on of enzyme when ATP/ADP ra=o is high iii) Phosphoryla=on inhibited by ADP and pyruvate Isocitrate Dehydrogenase •  Allosteric ac=va=on/inhibi=on by ADP/ATP Alpha ketoglutarate dehydrogenase •  Inhibited by high Sucinyl CoA, ATP and NADH Your Learning From Today Should focus on being able to;
•  Outline how pyruvate generated in glycolysis is fed into the KCAC chain of enzymes in the mitochondrial matrix •  Relate the events occurring in the KCAC to a meaningful biological impera=ve, that is the harves=ng of high energy electrons for use in driving ATP synthesis •  List and describe the enzyma=c reac=ons occurring at each step of Kreb’s Citric Acid Cycle (KCAC). (substrates, enzymes, products, reac=on types) •  Discuss allosteric checkpoints in the KCAC