Biochemistry Ch 9 135-150
Regulation of Enzymes – type of regulation reflects function for the pathway and need for that pathway
in particular tissue or cell
-pathways that produce a product are usually feedback regulated (allosteric inhibition, or
induction/suppression)
-Storage and toxic disposal pathways are regulated by feed-forward mechanisms that reflects availability
of precursor
Regulation can be organized into 3 categories: reversible binding to active site, regulation by changing
conformation of active site (allosteric inhibition), and regulation by changing concentration of enzyme
Regulation by Substrate and Product Concentration – velocity of all enzymes is dependent on
concentration of substrate, reflected in starvation in which several pathways are deprived of substrate.
-storage and toxic waste disposal pathways normally regulated to speed up when more substrate is
available
Michaelis-Menten Equation – equations of enzyme kinetics provide quantitative way to describe
dependence of enzyme on substrate concentration
-Michaelis-Menten Equation relates initial velocity to the concentration of substrate and the parameters
Km and Vmax (maximum velocity that can be achieved at an infinite substrate concentration)
-Km is the concentration of substrate required to reach 1/2Vmax
Where Km = (Kr + Kcat)/kf and Vmax = Kcat + E
Saturation Kinetics – point at Vmax where it cannot go any faster because enzyme is saturated
with substrate
-velocity of an enzyme is most sensitive to changes in substrate concentration below the Km
-Km of an enzyme is related to the dissociation constant
-The higher the Km, the more substrate is needed to reach 1/2Vmax, therefore a mutation that alters
binding site will increase Km.
Hexokinase Isozymes have Different Km Values for Glucose – hexokinase in red blood cells and in the
liver is different
-Hexokinase converts glucose to G-6-P by hydrolysis of ATP. G6P can go into glycolysis or be converted
to glycogen
-Hexokinase I is in red blood cells has a Km of 0.05mM
-Glucokinase (the hexokinase in liver and pancreas) has Km of 5 to 6 mM
-RBC is dependent only on glucose metabolism for its ATP, so the low Km means that blood
glucose can drop to a low level and the enzyme would still be able to phosphorylate
-Since the liver stores a large amount of excess glucose as glycogen or converts it to fat, the rate
of phosphorylation in the liver will tend to increase as blood glucose increases after a high-carb
meal and decreases as blood glucose falls. High Km promotes storage of glucose as liver
glycogen but only when glucose is in excess supply
Velocity and Enzyme Concentration – Rate of a reaction is directly proportional to concentration of
enzyme, if you double the enzyme you double the amount of product formed.
Multisubstrate Reactions – Most enzymes have more than one substrate, and the substrate-binding
sites overlap in the catalytic site, therefore the apparent Km depends on concentration of cosubstrate or
product
Rates of Enzyme-Catalyzed Reactions in the Cell – Although many physiological enzymes deviate from
michaelis menten due to more than one substrate and other factors, the term Km is still used for these
enzymes to approximate concentration at which velocity = 1/2Vmax
Reversible Inhibition within the Active Site – can alter enzyme activity by binding compounds to the
active site
-Inhibitor is a compound that decreases the velocity of a reaction by binding to an enzyme, it is
reversible if it can dissociate at a significant rate. Reversible inhibitors are separated into competitive
inhibitors, noncompetitive, or uncompetitive
1.) Competitive Inhibition – this inhibitor competes with substrate for binding at the active site and is
usually a close structural analog to the substrate, can be overcome with an increase in substrate
concentration, competitive inhibitors increase value of Km
2.) Noncompetitive Inhibitors – does not compete with substrate for its binding site. If you have two
susbtrates that bind to enzyme A and B, but an inhibitor is structurally related to B and binds in its place,
it is non-competitive with respect to A
-noncompetitive inhibitors will lower concentration of enzyme and lower Vmax, but not lower
Km for A
-can be metals, might not bind at either substrate-recognition site
Simple Product Inhibition in Metabolic Pathways – all products are reversible inhibitors of enzymes
that produce them and may be competitive or noncompetitive
-Simple product inhibition is when product decreases the rate of an enzyme
-in metabolic pathways, this prevents one enzyme in a sequence of reactions from generating too much
product faster than it can be used by the next enzyme
-for example, G6P inhibits hexokinase to conserve blood glucose for tissues that need it
Regulation Through Conformational Changes – Rate of reaction is affected by binding of substrate to its
catalytic site. Changing the conformation of the enzyme can affect the catalytic site. This can include:
allosteric activation and inhibition, phosphorylation or covalent modification, protein-protein
interactions, and proteolytic cleavage
Conformation Changes in Allosteric Enzymes – allosteric activators and inhibitors bind to an allosteric
site (not the active site) and cause conformational change in the enzyme that affects the affinity of
enzyme to substrate
Cooperativity in Substrate Binding to Allosteric Enzymes – allosteric enzymes contain two or more
subunits and exhibit positive cooperativity – binding of one substrate to a subunit helps binding of a
second substrate to another subunit
-first substrate has difficulty binding, because enzyme is in the “Taut” conformation.
-once bound, that substrate changes affinity of an adjacent subunit to make it high-affinity
-hemoglobin is an example tetramer, change in 1 subunit affects all subunits
Allosteric Activators and Inhibitors – binding of an allosteric activator changes the catalytic site so that
it increases affinity of enzyme for substrate
-activators bind more tightly to the R (open) state than closed (“taut”) enzyme. Thus, activators increase
amount of enzyme in the active state
-inhibitors bind more tightly to the T state than the R state, so that substrate or activator must be
increased to overcome the effects of the allosteric inhibitor
-Allosteric inhibitors can have their own binding site or compete for substrate at active site (can be a
term to be applied to any inhibitor of allosteric enzyme)
-without activator, plot of velocity vs substrate results in an S-shaped curve – whereas with activator,
the plot looks more hyperbolic with substantial decrease in Km
-these activators are called K effectors, because they change Km but not Vmax
-inhibitors shift the curve to the right and increase Km or increasing Km and decreasing Vmax
-Examples: glycogen phosphorylase, phosphofructokinase-1, isocitrate dehydrogenase are all allosteric
enzymes regulated by concentrations of ADP and AMP, which are allosteric activators. When ATP drops,
ADP and AMP increase, inducing these enzymes
Allosteric Enzymes in Metabolic Pathways – allosteric inhibitors have a much stronger effect on enzyme
velocity than competitive and noncompetitive inhibitors in an active catalytic cycle
-Allosteric effector is rapid, occurring as soon as changes in concentration occurs, essential for feedback
-Allosteric effectors need not resemble substrates
Conformation Changes from Covalent Modification
1.) Phosphorylation – many enzymes are regulated through phosphorylation by a protein kinase or
dephosphorylation by a phosphatase
a. Serine/threonine kinases transfer PO4 from ATP to a serine or threonine on enzyme
b. Tyrosine kinase transfer PO4 to the hydroxyl on the tyrosine residue
-phosphate is bulky, causes conformational change at the catalytic site which makes some
enzymes more active and some enzymes less active
2.) Muscle Glycogen Phosphorylase – rate limiting enzyme in the pathway of glycogen degradation,
degrades glycogen to glucose 1-phosphate
a. regulated by allosteric activator AMP, which increases as ATP is used for muscular
contraction
b. glycogen degradation is increased when an increase in AMP signals more fuel is needed
c. Can be activated by phosphorylation (glycogen phosphorylase kinase), either
phosphorylation or AMP binding can change the enzyme to the fully active
conformation
d. Phosphate is removed by protein phosphatase-1.
e. Phosphorylase kinase links phosphorylase to changes in level of adrenaline in blood,
regulated through phosphorylation by protein kinase A and by Ca2+ - Calmodulin during
contraction
3.) Protein Kinase A – some kinases are tightly bound to a single protein and regulate only the
protein to which they are bound, while others are non-specific to one enzyme
a. Protein kinase A is a serine/threonine kinase that phosphorylates several enzymes that
regulate different metabolic pathways (such as glycogen phosphorylase kinase)
b. Provides a means for hormones to control metabolic pathways
i. Adrenaline increases intracellular cAMP which is an allosteric second
messenger. cAMP binds to regulatory subunits of protein kinase A, which
dissociate and release the active subunits
Other Covalent Modifications – several proteins can have acetyl groups, ADP-ribose, or lipids, which can
all either activate or inhibit an enzyme
Conformational Changes from Protein-Protein Interactions – active site conformation can be
influenced by protein-protein interactions, such as with CA2+ - calmodulin and small G proteins
1. Calcium-Calmodulin Family of Proteins – modulator proteins bind to other proteins and
regulate their activity by causing conformational change at catalytic site or blocking it
a. Calcium calmodulin is an example of a dissociable modulator protein that binds to
several different proteins and regulates their function
b. Muscle glycogen phosphorylase kinase is activated by calcium-calmodulin in addition to
protein kinase A
i. Nerve impulse triggers calcium release from sarcoplasmic reticulum, Ca binds to
calmodulin on muscle glycogen phosphorylase kinase -> conformation change
which activates glycogen phosphorylase
ii. Ca also binds to troponin C
2. Small G proteins Regulate through conformational changes – small single subunit proteins that
bind and hydrolyze GTP. Binding changes the protein’s conformation enabling it to bind to a
target protein which is then activated or inhibited
-G proteins are GTPases that slowly hydrolyze their own GTP to GDP and phosphate, which
changes their conformation and disassembles them from target protein.
-their bound GDP is replaced by GTP and the process can start over again
-G-proteins are regulated by accessory proteins GAPS, GEFS, and GDIs, which can also be
regulated by allosteric effectors
a. GAPS (GTPase-activating proteins) – increase rate of GTP hydrolysis by G protein and
rate of G protein-target protein dissociation
b. GEFs (guanine nucleotide exchange factor) – when it binds to g protein, it increases
rate of GTP/GDP exchange, activing the G protein
c. GDI (GDP dissociation Inhibitors) – bind to GDP-G protein and inhibit dissociation of
GDP, inactivating the G-protein
-Ras superfamily of G-proteins dividied into Ras, Rho, Arf, Rab, and Ran.
-these all regulate cell growth, morphogenesis, cell motility, axon guidance, cytokinesis
Proteolytic Cleavage – enzymes can undergo cleavage during synthesis in the cell, or be packaged by
lysosomes or secretory vesicles and excreted from the cell as proenzymes (precursor proteins that
undergo cleavage to become fully functional)
-proteolytic cleavage is irreversible
-precursors to proteases are called zymogens, which need cleavage so that they don’t become active too
early
-Chymotrypsinogen is a zymogen that is stored in vesicles in pancreatic cells until secreted into intestinal
lumen via the major duodenal papilla
-converted to chymotrypsin through cleavage by trypsin
Regulation Through Changes in Amount of Enzyme – tissues always adjust rate at which enzyme is
synthesized. Vmax considers the rate of reaction is proportional to the amount of enzyme present
1. Regulated Enzyme Synthesis – enzymes can be induced or repressed, or mRNA can be stabilized
2. Regulated Protein Degradation – content of an enzyme in the cell can be altered through
selective regulated degradation as well as through regulated synthesis
-degradation happens through proteasomes and caspases
-during fasting, protein degradation in skeletal muscle is activated to sincrease supply of
amino acids in blood for gluconeogenesis
-ubiquitin targets proteins to proteasomes is increased by cortisol
Regulation of Metabolic Pathways – different pathways have various methods of regulating enzyme
activity
1. Regulation Occurs at Rate Limiting Step – pathways are generally regulated by one enzyme,
called the regulatory enzyme which is the rate-limiting reaction of the pathway
a. this is the slowest step and influences the rest of pathway
b. additional regulated enzymes occur at branch points in pathway
c. inhibition of a rate limiting enzyme in a pathway leads to accumulation of the pathway
precursor
2. Feedback Regulation – end product of a pathway controls its own rate of synthesis, involves
allosteric regulation of the rate-limiting enzyme by the end product of a pathway
a. Can control its own synthesis by inducing or repressing the gene for synthesis – much
slower to respond to changing conditions than allosteric regulation
3. Feed-forward Regulation – disposal of toxic compounds are feed-forward regulated
a. may occur through an increased supply of substrate to an enzyme with a high Km,
allosteric activation of rate-limiting enzyme, substrate-related induction of gene
expression, or increased concentration of hormone that stimulates a storage pathway
4. Tissue isozymes of Regulatory Proteins – different tissue types only synthesize proteins that
they require, meaning same enzymes in different tissues may have different regulatory
pathways, such as glucokinase and hexokinase
5. Counterregulation of Opposing Pathways – a pathway for synthesis usually has one or more
enzymatic steps that differ from the pathway for degradation of the compounds
a. glycogen synthesis is activated when glycogen degradation is inhibited
6. Substrate Channeling through Compartmentation – compartmentation of enzymes in
multienzyme complexes provides a means for regulation, such as all enzymes of TCA cycle are in
mitochondrion, allowing the sequential reactions to take place irrespective of cytoplasmic
environment
a. Similarly, multienzyme complexes allow a substrate to be converted to a product and
move in an assembly-line fashion straight to the next active site to be converted once
again
Disorders
1. Alcoholism – alcohol dehydrogenase and microsomal ethanol oxidizing system are actively
detoxifying alcohol. High NADH can inhibit alcohol dehydrogenase, allowing toxic metabolites
to accumulate
2. Anorexia – Effects of malnutrition on energy production was discussed
3. Maturity onset diabetes of the young – mutations in various proteins can lead to this,
specifically pancreatic glucokinase