11/4/2010enzyme mechanisms & regulation enzymes v: specific mechanisms; regulation andy howard...
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11/4/2010Enzyme Mechanisms & Regulation
Enzymes V:Specific
Mechanisms; Regulation
Andy HowardIntroductory Biochemistry
4 November 2010
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Examples of mechanisms
We’ll finish our detailed look at the serine protease mechanism, and then explore a few other mechanisms to illustrate specific ideas
Then we’ll begin our discussion of regulation of enzymes
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Mechanisms and Regulation Serine Proteases
Chymotrypsin Evolution
Other mechanisms Cysteinyl proteases
Lysozyme TIM
Regulation by thermodynamics
Enzyme availability Transcription Degradation Compartmentation
Allostery Mechanisms Kinetics PTM
Hemoglobin & myoglobin as instances
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Modes of catalysis in serine proteases Proximity effect: gathering of reactants in steps 1 and 4
Acid-base catalysis at histidine in steps 2 and 4
Covalent catalysis on serine hydroxymethyl group in steps 2-5
So both chemical (acid-base & covalent) and binding modes (proximity & transition-state) are used in this mechanism
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What mechanistic concepts do serine proteases not illustrate? Quaternary structural effects(We’ll discuss this under regulation…)
Protein-protein interactions(Becoming increasingly important)
Allostery(also will be discussed under regulation)
Noncompetitive inhibition
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Specificity Active site catalytic triad is nearly invariant for eukaryotic serine proteases
Remainder of cavity where reaction occurs varies significantly from protease to protease.
In chymotrypsin hydrophobic pocket just upstream of the position where scissile bond sits
This accommodates large hydrophobic side chain like that of phe, and doesn’t comfortably accommodate hydrophilic or small side chain.
Thus specificity is conferred by the shape and electrostatic character of the site.
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Chymotrypsin active site Comfortably accommodates aromatics at S1 site
Differs from other mammalian serine proteases in specificity
Diagram courtesy School of Crystallography, Birkbeck College
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Divergent evolution Ancestral eukaryotic serine proteases presumably have differentiated into forms with different side-chain specificities
Chymotrypsin is substantially conserved within eukaryotes, but is distinctly different from elastase
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iClicker quiz, question 1:Why are proteases often synthesized as zymogens? (a) Because the transcriptional machinery cannot function otherwise
(b) To prevent the enzyme from cleaving peptide bonds outside of its intended realm
(c) To exert control over the proteolytic reaction
(d) None of the above
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Convergent evolution Reappearance of ser-his-asp triad in unrelated settings
Subtilisin: externals very different from mammalian serine proteases; triad same
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Subtilisin mutagenesis
Substitutions for any of the amino acids in the catalytic triad has disastrous effects on the catalytic activity, as measured by kcat.
Km affected only slightly, since the structure of the binding pocket is not altered very much by conservative mutations.
An interesting (and somewhat non-intuitive) result is that even these "broken" enzymes still catalyze the hydrolysis of some test substrates at much higher rates than buffer alone would provide. I would encourage you to think about why that might be true.
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Cysteinyl proteases Ancestrally related to ser proteases?
Cathepsins, caspases, papain
Contrasts: Cys —SH is more basicthan ser —OH
Residue is less hydrophilic
S- is a weaker nucleophile than O-
Diagram courtesy ofMariusz Jaskolski,U. Poznan
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Papain active site
Diagram courtesy Martin Harrison,Manchester University
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Hen egg-white lysozyme Antibacterial protectant ofgrowing chick embryo
Hydrolyzes bacterial cell-wall peptidoglycans
“hydrogen atom of structural biology” Commercially available in pure form Easy to crystallize and do structure work Available in multiple crystal forms
Mechanism is surprisingly complex (14.7)
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
HEWLPDB 2vb1
0.65Å15 kDa
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Mechanism of lysozyme
Strain-induced destabilization of substrate makes the substrate look more like the transition state
Long arguments about the nature of the intermediates
Accepted answer: covalent intermediate between D52 and glycosyl C1 (14.39B)
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The controversy
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Triosephosphate isomerase(TIM) dihydroxyacetone phosphate glyceraldehyde-3-phosphate
Km=10µMkcat=4000s-1
kcat/Km=4*108M-1s-1
DHAP
Glyc-3-P
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TIM mechanism DHAP carbonyl H-bonds to neutral imidazole of his-95; proton moves from C1 to carboxylate of glu165
Enediolate intermediate (C—O- on C2)
Imidazolate (negative!) form of his95 interacts with C1—O-H)
glu165 donates proton back to C2 See Fort’s treatment (http://chemistry.umeche.maine.edu/CHY431/Enzyme3.html)
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Enzymes are under several levels of control
Some controls operate at the level of enzyme availability
Other controls are exerted by thermodynamics, inhibition, or allostery
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Regulation of enzymes The very catalytic proficiency for which enzymes have evolved means that their activity must not be allowed to run amok
Activity is regulated in many ways: Thermodynamics Enzyme availability Allostery Post-translational modification Protein-protein interactions
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Thermodynamics as a regulatory force Remember that Go’ is not the determiner of spontaneity: G is.
Therefore: local product and substrate concentrations determine whether the enzyme is catalyzing reversible reactions to the left or to the right
Rule of thumb: Go’ < -20 kJ mol-1 is irreversible
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Enzyme availability
The enzyme has to be where the reactants are in order for it to act
Even a highly proficient enzyme has to have a nonzero concentration
How can the cell control [E]tot? Transcription (and translation) Protein processing (degradation) Compartmentalization
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Transcriptional control mRNAs have short lifetimes
Therefore once a protein is degraded, it will be replaced and available only if new transcriptional activity for that protein occurs
Many types of transcriptional effectors Proteins can bind to their own gene Small molecules can bind to gene Promoters can be turned on or off
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Protein degradation All proteins havefinite half-lives;
Enzymes’ lifetimes often shorter than structural or transport proteins
Degraded by slings & arrows of outrageous fortune; or
Activity of the proteasome, a molecular machine that tags proteins for degradation and then accomplishes it
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Compartmentalization
If the enzyme is in one compartment and the substrate in another, it won’t catalyze anything
Several mitochondrial catabolic enzyme act on substrates produced in the cytoplasm; these require elaborate transport mechanisms to move them in
Therefore, control of the transporters confers control over the enzymatic system
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Allostery Remember we defined this as an effect on protein activity in which binding of a ligand to a protein induces a conformational change that modifies the protein’s activity
Ligand may be the same molecule as the substrate or it may be a different one
Ligand may bind to the same subunit or a different one
These effects happen to non-enzymatic proteins as well as enzymes
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Substrates as allosteric effectors (homotropic) Standard example: binding of O2 to one subunit of tetrameric hemoglobin induces conformational change that facilitates binding of 2nd (& 3rd & 4th) O2’s
So the first oxygen is an allosteric effector of the activity in the other subunits
Effect can be inhibitory or accelerative
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Other allosteric effectors (heterotropic) Covalent modification of an enzyme by phosphate or other PTM molecules can turn it on or off
Usually catabolic enzymes are stimulated by phosphorylation and anabolic enzymes are turned off, but not always
Phosphatases catalyze dephosphorylation; these have the opposite effects
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Cyclic AMP-dependent protein kinases
Enzymes phosphorylate proteins with S or T within sequence R(R/K)X(S*/T*)
Intrasteric control:regulatory subunit or domain has a sequence that looks like the target sequence; this binds and inactivates the kinase’s catalytic subunit
When regulatory subunits binds cAMP, it releases from the catalytic subunit so it can do its thing
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Kinetics of allosteric enzymes Generally these don’t obey Michaelis-Menten kinetics
Homotropic positive effectors produce sigmoidal (S-shaped) kinetics curves rather than hyperbolae
This reflects the fact that the binding of the first substrate accelerates binding of second and later ones
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T R State transitions Many allosteric effectors influence the equilibrium between two conformations
One is typically more rigid and inactive, the other is more flexible and active
The rigid one is typically called the “tight” or “T” state; the flexible one is called the “relaxed” or “R” state
Allosteric effectors shift the equilibrium toward R or toward T
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MWC model for allostery Emphasizes
symmetry and symmetry-breaking in seeing how subunit interactions give rise to allostery
Can only explain positive cooperativity
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Koshland (KNF) model Emphasizes conformational changes from one state to another, induced by binding of effector
Ligand binding and conformational transitions are distinct steps
… so this is a sequential model for allosteric transitions
Allows for negative cooperativity as well as positive cooperativity
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Heterotropic effectors
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Post-translational modification We’ve already looked at phosphorylation
Proteolytic cleavage of the enzyme to activate it is another common PTM mode
Some proteases cleave themselves (auto-catalysis); in other cases there’s an external protease involved
Blood-clotting cascade involves a series of catalytic activations
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Zymogens As mentioned earlier, this is a term for an inactive form of a protein produced at the ribosome
Proteolytic post-translational processing required for the zymogen to be converted to its active form
Cleavage may happen intracellularly, during secretion, or extracellularly
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Blood clotting
Seven serine proteases in cascade Final one (thrombin) converts fibrinogen to fibrin, which can aggregate to form an insoluble mat to prevent leakage
Two different pathways: Intrinsic: blood sees injury directly Extrinsic: injured tissues release factors that stimulate process
Come together at factor X
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Cascade
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Protein-protein interactions One major change in biochemistry in the last 20 years is the increasing emphasis on protein-protein interactions in understanding biological activities
Many proteins depend on exogenous partners for modulating their activity up or down
Example: cholera toxin’s enzymatic component depends on interaction with human protein ARF6
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Globins as aids to understanding Myoglobin and hemoglobin are well-understood non-enzymatic proteins whose properties help us understand enzyme regulation
Hemoglobin is described as an “honorary enzyme” in that it “catalyzes” the reactionO2(lung) O2 (peripheral tissues)
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Setting the stage for this story
Myoglobin is a 16kDa monomeric O2-storage protein found in peripheral tissues
Has Fe-containing prosthetic group called heme; iron must be in Fe2+ state to bind O2
It yields up dioxygen to various oxygen-requiring processes, particularly oxidative phosphorylation in mitochondria in rapidly metabolizing tissues
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Why is myoglobin needed? Free heme will bind O2 nicely;why not just rely on that?
Protein has 3 functions: Immobilizes the heme group Discourages oxidation of Fe2+ to Fe3+
Provides a pocket that oxygen can fit into
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Setting the stage II Hemoglobin (in vertebrates, at least) is a tetrameric, 64 kDa transport protein that carries oxygen from the lungs to peripheral tissues
It also transports acidic CO2 the opposite direction
Its allosteric properties are what we’ll discuss
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Structure determinations Myoglobin & hemoglobin were
the first 2 proteins to have their 3-D structures determined experimentally Myoglobin: Kendrew, 1958 Hemoglobin: Perutz, 1958 Most of the experimental tools that crystallographers rely on were developed for these structure determinations
Nobel prizes for both, 1965(small T!)
QuickTime™ and a decompressor
are needed to see this picture.
QuickTime™ and a decompressor
are needed to see this picture.
Photo courtesyOregon State Library
Photo courtesyEMBL