Enzyme Kinetics Lecture

When all enzyme, [Et] is [ES], V1 = Vmax = k2[Et], when all the enzyme is
saturated and completely working at full efficiency
V1 = Fraction ES * Vmax
V0 = [ES]/ ([E]+[ES])*Vmax
For all intents and purposes, Km = dissociation constant. Km = [E][S]/[ES]
[ES]=[E][S]/Km ; Plug this back in to get
V = ([S]/[S]+KM)
LINEWEAVER-BURKE- 1/v = Km/Vmax (1/s) + (1/Vmax)
Km = concentration of [S] where E is saturated
Km = Kd of MM
Turnover number = catalytic rate constant (Kcat)
o Vmax = Kcat (Et)
Kcat = Vmax / [Et]

Enzyme Regulation
Enzyme control
Allosteric control
Produce different isozymes
Covalent modification
Proteolytic cleavage make a zymogen (inactive form that can be turned on)
Control of amount
o Transcription/translation
Allostery aspartate transcarbamoylase (ATCase)
First step in pyrimidine biosynthesis
Active enzyme has 12 subunits (C6r6); C = catalytic; r = regulatory
o 3 on top and 3 on bottom
Sigmoidal (Cooperative) (like hemoglobin); cannot analyze like MM which is
more myoglobin-like
T and R states are MM-like
T is low activity, R is high activity state
If we remove the r chains, the activity resembles the C state (break it and it
works better lol); sigmoidal cooperativity is lost
Regulatory Chain
o No activity by itself
o r chain binds CTP
Regulatory chains bind CTP
CTP decreases activity of intact ATCase feedback inhibition
o CTP builds up and shuts down production of itself
Based on feedback inhibition of ATCase, activity is enhanced by ATP to make
sure that there is an equilibrium purines and pyrimidines

When pyr is in excess, ATCase is shut off; when purine is in excess, ATCase
activity is turned on
R chain binds CTP and ATP; allowing for double regulation of the enzyme by
both ATP and CTP
ATP bound- R state (binds w/ higher affinity to R state); CTP bound T state
CTP bound state is T state; More compact; Active site is less accessible
Aspartate binds more tightly to the R state (kind of like oxygen binding
better to the R state)
Isozymes
Result from gene duplication; lactate dehydrogenase (functional tetramer)
In heart
o Low Km; allosterically controlled (tunable)
Muscle
o High Km; No allosteric control
Intermediate tetramers (mixed copies) also have intermediate properties
Enzyme changes as you go from small to adult; Different temporal and tissue
distributions
Covalent Modifications
Attachment of chemical groups (mostly reversible)
Done by enzymes
Phosphorylation Benefits
-2 Charge Added for electrostatic interactions
Phosphate group can also hydrogen bond (HB acceptor)
Effects can be amplified if we regulate kinases/phosphatases
ATP is cellular energy supply; tie your activity to metabolic state
~30% of proteins regulated via phosphorylation
Phosphorylation properties
Phosphate bond energy ~25 kJ/mole
Switch-like behavior
Glycogen phosphorylase
Catalyzes first step in glycogen metabolism
Functions as a dimer
T state and R state (low and high affinities respectively)
Unphosphorylated T state favored; already lots of energy in cell
ATP and AMP allosterically regulated
Response changed in response to fear; activation of the sympathetic system
Phosphorylated R state is favored; eliminates allosteric control
Combination of allostery and modification

Glycolysis
Happens in the cytosol of cells
Converted to glycogen when too much ATP inside cell
Glucose needs to be imported
Uses glucose importers
ATP
Gamma phosphate is cleaved off to become an inorganic phosphate (Pi)
(through hydrolysis) releasing 7kcal/molecule ATP
Steps
1. Glucose enters and gets phosphorylated
a. Phosphorylated by hexokinase at the hydroxyl group of C6
b. Side note on kinases
i. Any xxxx kinase means that it typically uses ATP to
phosphorylate xxxx
c. Allows for glucose to be destabilized and kept inside the cell (size)
d. Net ATP count: -1
2. Conversion of gluose-6-phosphate into fructose-6-phosphate through
phosphoglucose isomerase
3. Conversion of isomer into fructose 1, 6-bisphosphate
a. Consumes 1 ATP
b. Conducted by PFK
c. Net ATP count: -2
4. Fructose 1, 6-bisphoate cleaved into two trioses by aldolate
a. Glyceraldehyde-3-phosphate
b. Dihydroxyacetone phosphate
5. Isomerase almost always converts it between the two
a. Side note
i. Diffusion controlled limit
1. Rate determined by how often they can possibly meet;
essentially a rxn happens every time they do (~10^8
M^-1 sec^-1)
6. First 5 Steps Summary
a. Glucose goes to glucose 6 phosphate through hexokinase
b. Isomerase converts it to fructose 1 phosphate
c. PFK converts that into fructose 1,6-biphosphate
d. Aldolase cleaves it into glyceraldehyde-3-phosphate and
dihydroxyacetone phosphate
e. Isomerase converts between two using diffusion controlled limit
f. Net ATP: -2 used in step 1 and in step 3
g. 2 ATP produced per triose later on; net ATP at end = +2 ATP
1. Glyceraldehyde-3-phosphate dehydrogenase (GAP)
a. Inorganic phosphate is added directy
b. NAD+ NADH
c. NADH acts as a reducing agent

2. 1st ATP generated via phosophoglycerate kinase which removes a Pi from

one in the glycerate 1,3-bisphosphate
3. Making another smaller molecule
4. Enolate to make PEP
5. Changing PEP
a. This molecule is in enol form and wants to go back to the keto form
b. Has to shed the phospsphate
c. Does so to create ATP; making pyruvate in enol form which stabilizes
to keto form
Lactate formation
Fermentation
Formation of NAD+ from NADH and metabolism of Pyruvate into toxic
materials
No further energy gain
Not much oxygen, but lots of glucose
Yeast
o Ethanol formation
Humans
o Lactate dehydrogenase makes C1 of pyruvate chiral, giving sterochem
and, thus, lactate
NADH and NAD+ are vitamin B derived cofactors and are used as reducing
and oxidizing agents respectively; used in hydride transfer
In creation of pyruvate, protons are made and lactate couples w/ these
protons and removed from the cell to the liver for gluconeogenesis
Lactate removed from cell via MCT (Monocarboxylate transport)
PFK
Homotetramer
o 1 active site/ subunit
o 2 allosteric sites
ATP is a negative effector of this; too much ATPbinding to
enzymeglycolysis slowed down
Protons bind to PFK to inhibit and slow down glycolysis
Glycolysis too little
ATP goes down
AMP goes up and alleviates effect of ATP on PFK, stimulating rate again