Module 2 Flashcards
Mammals cannot synthesize all coenzymes from scratch
A tightly bound metal or coenzyme is a ________
An apoenzyme plus a prosthetic group is a ________ .
prosthetic group
holoenzyme
ΔGo = \_\_\_\_\_\_\_\_\_\_\_\_\_\_ ΔG'o = \_\_\_\_\_\_\_\_\_\_\_\_\_\_
standard free energy change (298 K, 1 atm, 1 M)
biochemical standard free energy change (pH 7)
Enzymes do not alter the equilibrium between Sand P only the _________ __ ___ _________
rate of the reaction
Just because ΔG’ois negative does not mean that the reaction will take place at a detectable rate
Even though ΔG’ois very negative sucrose is very stable
Catalysts enhance reaction rates by lowering _______ _______
activation energies
______ ______:any species along a reaction pathway with finite chemical lifetime(longer than a molecular vibration ~10-13seconds)
Reaction intermediates
Reaction equilibria are linked to ____ (standard free energy change)
Reaction rates are linked to ΔG‡ (activation energy)
ΔG’o
ΔG‡
K’eq = [P]/[S]
ΔG’o= -RT ln K’eq
R, 8.315 J/mol•K
T, absolute temp in K
Keq and ΔG’o relationship
First order reaction: __ = __ * __
Second order reacion: __ = __ __ __
V=k[S]
V=k[S1]{S2}
How do enzymes speed up the rate of reactions?
- formation of covalent interactions with amino acid side chains or bound metals or cofactors
- non-covalent interactions (formation of EScomplex)
binding energy, ΔGB,is a major source of free energy used by enzymes to lower the activation energy of reactions
Enzyme active sites are complimentary to the ______ ______ of the reaction
transition state
Stronger/additional interactions with the transition state as compared to the ground state lower the ______ ______
activation barrier
Physical and thermodynamic factors contributing to ΔG‡:
1. ________________________
- ________________________
- ________________________
- ________________________
- reduction in entropy (less free motion)
- solvation shell of hydrogen-bonded water around Sand P
- distortion of substrates (like stickasedid)
- need for proper alignment of catalytic functional groups
______ _____ are the most common biochemical reactions
Proton transfers
In enzyme kinetics ____ ____ must be short so that [S] does not change much
Reaction times
___ is the substrate concentration at ½ Vmax
Km
enzyme is saturated with substrate at __
Vmax
- binding of substrate to enzyme
equilibrium is normally reached within microseconds
as [S] increases eventually the enzyme becomes saturated
- reaction and release of product
this is the slow step and limits the rate of the whole reaction
Reactions kinetics are dictated by a rapid, concentration-dependent binding of substrate to the enzyme followed by a slower generation of released product
Steady State Kinetics
Steady State Kinetics
- binding of substrate to enzyme
_____________________________
_____________________________
- reaction and release of product
_____________________________
_______ ______ are dictated by a rapid, concentration-dependent binding of substrate to the enzyme followed by a slower generation of released product
- equilibrium is normally reached within microseconds
as [S] increases eventually the enzyme becomes saturated
- this is the slow step and limits the rate of the whole reaction
Reactions kinetics
The ____-____ equation was based on a specific kinetic model but many enzymes that use different (complex) mechanisms yield a similar hyperbolic V vs [S]curve and the Vmax and Km’s determined are valuable parameters.
Michaelis-Menten
The turnover number, ___
kcat
If the rate limiting step is binding of substrate: kcat= ___
k1
If the rate limiting step is release of product: kcat = __
k3
If there are several slow steps kcat is a _______ function
complex
In all cases ____ is a first order rate constant (1/sec) and is equal to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate
kcat
parallel lines indicate a___-___ (double-displacement) pathway
Km for S1increases as [S2] increases
Ping-Pong
Inhibitor blocks binding of substrate
Vmaxis not affected by inhibitor, but the apparent Kmis raised
Usually inhibitor is not metabolized by the enzyme it blocks
Methanol is converted by alcohol dehydrogenase to toxic formaldehyde
Ethanol is like a competitive inhibitor (converted to acetaldehyde)
Reversible Competitive Inibition
Inhibitor interacts with ES at a site distinct from the active site and slows the reaction
Vmaxand apparent Kmare lowered by the inhibitor
Reversible Uncompetitive Inhibition
Inhibitor interacts with E or ES at a site distinct from the active site and slows the reaction
Vmaxis lowered and apparent Kmraised (shown above) or lowered by the inhibitor
Reversible Mixed Inhibition
Inhibitors that bind covalently or very strongly and destroy a required functional group are ________
Irreversible
_______ inhibitors bind to the enzyme, make it part way through the reaction mechanism then block subsequent steps
These make good drugs
Suicide
Enzyme activity depends on __
pH
1.Identification of substrates, cofactors, products, regulators
2.Sequence of formation of enzyme bound reaction intermediates
3.Structure of each intermediate and transition state
4.Rates of interconversion between intermediates
5.Energy contributed by all reacting and interacting groups to intermediates and transition states
How many enzymes are understood at this level?
____
However, many details of the function of Chymotrypsin, Hexokinase, Enolase, and Lysozyme are known
What you need to know to fully understand a mechanism:
1. 2. 3. 4. 5.
None
________ specific for peptide bonds next to Trp, Phe,Tyr
Transition state stabilization, acid base catalysis
109fold stimulation of hydrolysis of peptide bond without using water
25 kDa protein cut in 2 places to produce 3 chains
Protease
Relative electronegativities
(attraction of electrons)
P N F O H S C
Nucleophile ______ ______
Electrophile ______ ______
F > O > N > C = S > P = H
(nucleus lover)
(electron lover)
What is important about the ___________ mechanism?
- Chymotrypsin is a serine protease that cleaves polypeptide backbone following F, Y or W residues because of hydrophobic pocket
- Activated by proteolytic cleavage
- Has a catalytic triad (Ser, His, Asp) in active site (D works through H to activate S)
- Uses acid base catalysis; general (Ser 195) and specific (H20)
- Uses covalent catalysis with N terminal portion of polypeptide attached
- Oxyanion hole stabilizes intermediates
- Pre-steady state kinetics gave evidence of a two step mechanism
chymotrypsin
You can fool hexokinase (kind of)
Xylose binds to hexokinase, but in a way that it can’t be phosphorylated
However, xylose increases the rate of hydrolysis of ATP
Xylose causes the hexokinase to enter it’s active conformation and tricks hexokinase into using water to hydrolyze the ATP
You can fool hexokinase (kind of)
Xylose binds to hexokinase, but in a way that it can’t be phosphorylated
However, xylose increases the rate of hydrolysis of ATP
Xylose causes the hexokinase to enter it’s active conformation and tricks hexokinase into using water to hydrolyze the ATP
Tools for examination of transition state complementarity
- Structure activity correlations
- Transition-state analogs
- Catalytic antibodies
- Structure activity correlations
If enzyme stabilizes the transition state then there must be interactions between substrate and the enzyme only in the transition state (this would not affect initial binding of substrate
2.Transition-state analogs
Compounds that mimic the structure thought to be present in the transition state
Because the enzyme usually aims to strain the substrate toward the transition state (to reduce activation energy) the binding of the analog is stronger than that of the substrate (100 to 1,000,000 times stronger)
Drugs such as those that inhibit
the HIV protease mimic the
transition state, but trap the
enzyme in an unproductive state
3.Catalytic antibodies
Antibodies are proteins that bind to specific structures
An antibody raised against a transition state analog might bind to a substrate and strain it to the transition state
Antibodies to phosphonate or phosphate esters act as enzymes to speed up hydrolysis of the ester or carbonate by 1000 to 10,000 fold
________ _______ needed to control complex pathways
usually the first enzyme of a pathway or branch point
Some enzymes are controlled by covalent modification (phosphorylation, adenylation etc.)
Regulatory enzymes
Most regulatory enzymes are _______ proteins
multimeric
_______ enzymes –conversion between active and inactive conformations controlled by reversible, noncovalent binding of regulatory compounds (allosteric modulators or effectors)
Allosteric
__________ __________
used in an early step in biosynthesis of pyrimidine nucleotides
12 subunits (6 regulatory, 6 catalytic)
Inactive in the _ state
Active in the _ state
Aspartate transcarbamoylase
T tensed
R relaxed
________ _______
(example: conversion of threonineto isolucine)
5 step pathway uses 5 enzymes
only E1 is regulated allosterically
Isoleucine binds to and inhibits E1
(end product inhibition)
E2-5 are not allosterically controlled by Isoleucine
controlling the first enzyme in a pathway ensures that potentially toxic intermediates don’t build up
_______ ______allows the level of the final product to control flux through the entire pathway so that a relatively constant steady state level of the final product is maintained
Feedback inhibition
Allosteric enzymes have a ______ curve instead of a hyperbolic curve found in Michaelis-Menten kinetics
Sigoidal
_______ curve results from cooperative interaction between subunits (like hemoglobin)
(a K0.5value replaces the Kmdesignation)
usually Vmaxis constant and K0.5is changed by effector
sigmoidal
Regulation by covalent modification
1. Functional group modifications: - - - - 2. Large molecule modifications: - - 3. Protein modification: - -
Phosphorylation
Acetylation
Adenylylation
Methylation
Myristoylation
ADP ribosylation
Ubiquitination
Sumolation
_______ ______ enzymes that phosphorylate proteins and
protein kinasesare
_______ _______remove the phosphate without regenerating ATP
protein phosphatases
_______-simple sugars (sakcharon -greek for sugar)
Monosaccharides
_______-2 monosaccharides connected by glycosidic bonds
Disaccharides
_______-short chains from 2 to 20 monosaccharides
Oligosaccharide
_______- polymer containing more than 20 monosaccharides
Polysaccharides
______ differ by configuration at only one chiral center
epimers
_______ interconversion of two hemiacetal forms
Mutarotation
Change in ______ requires breaking of covalent bond
configuration
Change in _______ requires breaking of covalent bond
conformation
Polysaccharides (glycans)
Most carbohydrates in nature are found in ________
Used as fuel storage (glycogen, starch)
Structural element (cellulose and chitin extracellular matrix)
Not usually of defined molecular weight like proteins
Characterized by type of monomer, length of chain, types of glycosidic bonds, degree of branching
polymers
Characterized by type of ____, ____ __ ____, ____ __ ____ ____, _____ __ ____.
monomer
length of chain
types of glycosidic bonds
degree of branching
Used as fuel storage (glycogen, starch)
Structural element (cellulose and chitin extracellular matrix)
Not usually of defined molecular weight like proteins
Characterized by type of monomer, length of chain, types of glycosidic bonds, degree of branching
Polysaccharides (glycans)
amylose
a form of starch
long, unbranched chains of D-glucose residues
amylopectin
another form of starch
branch every 24-30 residues
Which end are residues removed from when energy is needed?
Nonreducing end
____ is a multi-branched polysaccharide of glucose
Glycogen
glucose is stored with same linkages as that found in ______
amylopectin
_____ branching is more extensive (every 8-12 residues) 1,000,000 Mw
glycogen (glucose)
_____ is 7% of wet weight of liver and also found in skeletal muscle
glycogen (glucose)
each molecule of ____ has one reducing end and many non-reducing ends
enzymes remove glucose monomers from the non-reducing ends
glycogen (glucose)
________ store glucose in glycogen chains to reduce the osmolarity (otherwise there would be 0.4 M glucose)
Hepatocytes
Also it would be difficult to transport _____ into a liver cell up a gradient from 5 mM in the blood to 400 mM in the liver cell
glucose
_____ are bacterial and yeast polysaccharides
(α1 -> 6) linkage with (α1 -> 3) linkage at branch points
Dental plaque is ____ deposited by bacteria growing on teeth
dextrans
______ is found in stalks and stems and trunks -cotton is almost 100% ______
linear, unbranched homopolysaccharide 10,000 to 15,000 glucose units
Unlike amylose, amylopectin and glycogen _____ has βconfiguration
Most animals can’t digest cellulose (bacteria, fungi, and termites can)
cellulose
Hydrogen boning networks between stands of _____ provide fiber strength
cellulose
Fungi have ____
cellulase
_______ is a linear homopolysaccharide with N-acetylglucosamine (β linkage)
chitin
