Andy Howard Introductory Biochemistry, Spring 2008 19 February
2008
Enzyme Mechanisms Andy Howard Introductory Biochemistry, Spring
February 2008 Biochemistry: Enzyme Mechanisms 19 Feb 2008 How do
enzymes reduce activation energies?
We want to understand what is really happening chemically when an
enzyme does its job. Wed also like to know how biochemists probe
these systems. Biochemistry: Enzyme Mechanisms 19 Feb 2008
Mechanism Topics Terminology Binding Modes of Catalysis
Transition States Enzyme chemistry Diffusion-controlled Reactions
Binding Modes of Catalysis Induced-fit Tight Binding of Ionic
Intermediates Serine proteases Biochemistry: Enzyme Mechanisms 19
Feb 2008 Functional Note Henceforth we wont be providing as much
textbook-independent stuff Well rely more heavily on Hortons own
treatments If you dont understand something in Horton, ask me or
one of the other IIT biochemists Well provide clear syllabi for
exams Biochemistry: Enzyme Mechanisms 19 Feb 2008 Atomic-Level
Mechanisms
We want to understand atomic-level events during an enzymatically
catalyzed reaction. Sometimes we want to find a way to inhibit an
enzyme in other cases we're looking for more fundamental knowledge,
viz. the ways that biological organisms employ chemistry and how
enzymes make that chemistry possible. Biochemistry: Enzyme
Mechanisms 19 Feb 2008 How we study mechanisms
There are a variety of experimental tools available for
understanding mechanisms, including isotopic labeling of
substrates, structural methods, and spectroscopic kinetic
techniques. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Ionic
reactions Define them as reactions that involve charged, or at
least polar, intermediates Typically 2 reactants Electron rich
(nucleophilic) reactant Electron poor (electrophilic) reactant
Conventional to describe reaction as attack of nucleophile on
electrophile Drawn with nucleophile donating electron(s) to
electrophile Biochemistry: Enzyme Mechanisms 19 Feb 2008 Attack on
Acyl Group Transfer of an acyl group: scheme 6.1
Nucleophile Y attacks carbonyl carbon, forming tetrahedral
intermediate X- is leaving group Biochemistry: Enzyme Mechanisms 19
Feb 2008 Direct Displacement Attacking group adds to face of atom
opposite to leaving group (scheme 6.2) Transition state has five
ligands; inherently less stable than scheme 6.1 Biochemistry:
Enzyme Mechanisms 19 Feb 2008 Cleavage Reactions Both electrons
stay with one atom
Covalent bond produces carbanion: R3CH R3C:- + H+ Covalent bond
produces carbocation: R3CH R3C+ + :H- One electron stays with each
product Both end up as radicals R1OOR2 R1O + OR2 Radicals are
highly reactive some more than others Biochemistry: Enzyme
Mechanisms 19 Feb 2008 Oxidation-Reduction Reactions
Commonplace in biochemistry: EC 1 Oxidation is a loss of electrons
Reduction is the gain of electrons In practice, often: oxidation is
decrease in # of C-H bonds; reduction is increase in # of C-H bonds
Intermediate electron acceptors and donors are organic moieties or
metals Ultimate electron acceptor in aerobic organisms is usually
dioxygen (O2) Biochemistry: Enzyme Mechanisms 19 Feb 2008
Biological redox reactions
Generally 2-electron transformations Often involve alcohols,
aldehydes, ketones, carboxylic acids, C=C bonds: R1R2CH-OH + X
R1R2C=O + XH2 R1HC=O + X + OH- R1COO- + XH2 X is usually NAD, NADP,
FAD, FMN A few biological redox systems involve metal ions or Fe-S
complexes Usually reduced compounds are higher-energy than the
corresponding oxidized compounds Biochemistry: Enzyme Mechanisms 19
Feb 2008 Overcoming the barrier
Simple system: single high-energy transition state intermediate
between reactants, products Free Energy G R P Reaction Coordinate
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Intermediates Often
there is a quasi-stable intermediate state midway between reactants
& products; transition states on either side T2 T1 Free Energy
I R P Reaction Coordinate Biochemistry: Enzyme Mechanisms 19 Feb
2008 Activation energy & temperature
Its intuitively sensible that higher temperatures would make it
easier to overcome an activation barrier Rate k(T) = Q0exp(-G/RT) G
= activation energy or Arrhenius energy This provides tool for
measuring G Svante Arrhenius Biochemistry: Enzyme Mechanisms 19 Feb
2008 Determining G Remember k(T) = Q0exp(-G/RT) ln k = lnQ0 -
G/RT
Measure reaction rate as function of temperature Plot ln k vs 1/T;
slope will be -G/R catalyzed ln k uncatalyzed 1/T, K-1
Biochemistry: Enzyme Mechanisms 19 Feb 2008 How enzymes alter G
Enzymes reduce DG by allowing the binding of the transition state
into the active site Binding of the transition state needs to be
tighter than the binding of either the reactants or the products.
Biochemistry: Enzyme Mechanisms 19 Feb 2008 DG and Entropy Effect
is partly entropic:
When a substrate binds, it loses a lot of entropy. Thus the
entropic disadvantage of (say) a bimolecular reaction is soaked up
in the process of binding the first of the two substrates into the
enzyme's active site. Biochemistry: Enzyme Mechanisms 19 Feb 2008
Enthalpy and transition states
Often an enthalpic component to the reduction in DG as well Ionic
or hydrophobic interactions between the enzyme's active site
residues and the components of the transition state make that
transition state more stable. Biochemistry: Enzyme Mechanisms 19
Feb 2008 Two ways to change G Reactants bound by enzyme are
properly positioned Get into transition-state geometry more readily
Transition state is stabilized A B A B E E A+B A+B A-B A-B
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Reactive sidechains in
a.a.s
Group Functions Asp COO- -1 Cation binding, proton transfer Glu
Same as above His Imidazole ~0 Proton transfer Cys CH2SH Covalent
binding of acyl gps Tyr Phenol H-bonding to ligands Lys NH3+ +1
Anion binding, proton transfer Arg guanadinium Anion binding Ser
CH2OH See cys Biochemistry: Enzyme Mechanisms 19 Feb 2008
Generalizations about active-site amino acids
Typical enzyme has 2-6 key catalytic residues His, asp, arg, glu,
lys account for 64% Remember: pKa values in proteins sometimes
different from those of isolated aas Frequency overall Frequency in
catalysis Biochemistry: Enzyme Mechanisms 19 Feb 2008 Cleavages by
base : : Simple cleavage: XH + :B X:- + HB+
This works if X=N,O; sometimes C Removal of proton from H2O to
cleave C-X: O O O- COH + HN CN CN : : HO :B O HB+ H H :B
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Cleavage by acid
Covalent bond may break more easily if one of its atoms is
protonated Formation of unstable intermediate, R-OH2+, accelerates
the reaction Example:R+ + OH- ROH ROH2+ R+ + H2O (Slow) (Fast)
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Covalent catalysis
Reactive side-chain can be a nucleophile or an electrophile, but
nucleophile is more common AX + E XE + A XE + B BX + E Example:
sucrose phosphorylase Net reaction: Sucrose + Pi Glucose 1-P +
fructose Fructose=A, Glucose=X, Phosphate=B Biochemistry: Enzyme
Mechanisms 19 Feb 2008 Rates often depend on pH
If an amino acid that is necessary to the mechanism changes
protonation state at a particular pH, then the reaction may be
allowed or disallowed depending on pH Two ionizable residues means
there may be a narrow pH optimum for catalysis Biochemistry: Enzyme
Mechanisms 19 Feb 2008 Papain as an example Biochemistry: Enzyme
Mechanisms 19 Feb 2008 Diffusion-controlled reactions
Some enzymes are so efficient that the limiting factor in
completion of the reaction is diffusion of the substrates into the
active site: These are diffusion-controlled reactions. Ultra-high
turnover rates: kcat ~ 109 s-1. We can describe kcat / Km as
catalytic efficiency of an enzyme. A diffusion-controlled reaction
will have a catalytic efficiency on the order of 108 M-1s-1.
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Triosephosphate
isomerase (TIM)
dihydroxyacetone phosphate glyceraldehyde-3-phosphate Glyc-3-P DHAP
Km=10M kcat=4000s-1 kcat/Km=4*108M-1s-1 Biochemistry: Enzyme
Mechanisms 19 Feb 2008 TIM mechanism DHAP carbonyl H-bonds to
neutral imidazole of his-95; proton moves from C1 to carboxylate of
glu165 Enediolate intermediate (CO- on C2) Imidazolate (negative!)
form of his95 interacts with C1O-H) glu165 donates proton back to
C2 See Forts treatment or fig. 6.7. Biochemistry: Enzyme Mechanisms
19 Feb 2008 Examining enzyme mechanisms will help us understand
catalysis
Examining general principles of catalytic activity and looking at
specific cases will facilitate our appreciation of all enzymes
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Binding modes:
proximity
We describe enzymatic mechanisms in terms of the binding modes of
the substrates (or, more properly, the transition-state species) to
the enzyme. One of these involves the proximity effect, in which
two (or more) substrates are directed down potential-energy
gradients to positions where they are close to one another. Thus
the enzyme is able to defeat the entropic difficulty of bringing
substrates together. William Jencks Biochemistry: Enzyme Mechanisms
19 Feb 2008 Binding modes: efficient transition-state binding
Transition state fits even better (geometrically and
electrostatically) in the active site than the substrate would.
This improved fit lowers the energy of the transition-state system
relative to the substrate. Best competitive inhibitors of an enzyme
are those that resemble the transition state rather than the
substrate or product. Biochemistry: Enzyme Mechanisms 19 Feb 2008
Adenosine deaminase with transition-state analog
Transition-state analog: Ki~10-8 * substrate Km Wilson et al (1991)
Science 252: 1278 Biochemistry: Enzyme Mechanisms 19 Feb 2008
Induced fit Refinement on original Emil Fischer lock-and-key
notion:
both the substrate (or transition-state) and the enzyme have
flexibility Binding induces conformational changes Biochemistry:
Enzyme Mechanisms 19 Feb 2008 Example: hexokinase Glucose + ATP
Glucose-6-P + ADP
Risk: unproductive reaction with water Enzyme exists in open &
closed forms Glucose induces conversion to closed form; water cant
do that Energy expended moving to closed form Biochemistry: Enzyme
Mechanisms 19 Feb 2008 Hexokinase structure Diagram courtesy E.
Marcotte, UT Austin
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Tight binding of ionic
intermediates
Quasi-stable ionic species strongly bound by ion-pair and H-bond
interactions Similar to notion that transition states are the most
tightly bound species, but these are more stable Biochemistry:
Enzyme Mechanisms 19 Feb 2008 Serine protease mechanism
Only detailed mechanism that well ask you to memorize One of the
first to be elucidated Well studied structurally Illustrates many
other mechanisms Instance of convergent and divergent evolution
Biochemistry: Enzyme Mechanisms 19 Feb 2008 The reaction Hydrolytic
cleavage of peptide bond
Enzyme usually works on esters too Found in eukaryotic digestive
enzymes and in bacterial systems Widely-varying substrate
specificities Some proteases are highly specific for particular aas
at position 1, 2, -1, . . . Others are more promiscuous CH NH C NH
C NH R1 CH O Biochemistry: Enzyme Mechanisms R-1 19 Feb 2008
Mechanism Active-site serine OH Without neighboring amino acids,
its fairly non-reactive becomes powerful nucleophile because OH
proton lies near unprotonated N of His This N can abstract the
hydrogen at near-neutral pH Resulting + charge on His is stabilized
by its proximity to a nearby carboxylate group on an aspartate
side-chain. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Catalytic
triad The catalytic triad of asp, his, and ser is found in an
approximately linear arrangement in all the serine proteases, all
the way from non-specific, secreted bacterial proteases to highly
regulated and highly specific mammalian proteases. Biochemistry:
Enzyme Mechanisms 19 Feb 2008 Diagram of first three steps
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Diagram of last four
steps
Diagrams courtesy University of Virginia Biochemistry: Enzyme
Mechanisms 19 Feb 2008 Chymotrypsin as example
Catalytic Ser is Ser195 Asp is 102, His is 57 Note symmetry of
mechanism: steps read similarly L R and R L Diagram courtesy of
Anthony Serianni, University of Notre Dame Biochemistry: Enzyme
Mechanisms 19 Feb 2008 Oxyanion hole When his-57 accepts proton
from Ser-195: it creates an RO- ion on Ser sidechain In reality the
Ser O immediately becomes covalently bonded to substrate carbonyl
carbon, moving - charge to the carbonyl O. Oxyanion is on the
substrate's oxygen Oxyanion stabilized by additional interaction in
addition to the protonated his 57: main-chain NH group from gly 193
H-bonds to oxygen atom (or ion) from the substrate, further
stabilizing the ion. Biochemistry: Enzyme Mechanisms 19 Feb 2008
Oxyanion hole cartoon Cartoon courtesy Henry Jakubowski, College of
St.Benedict / St.Johns University Biochemistry: Enzyme Mechanisms
19 Feb 2008 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 Biochemistry: Enzyme
Mechanisms 19 Feb 2008 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 doesnt comfortably
accommodate hydrophilic or small side chain. Thus specificity is
conferred by the shape and electrostatic character of the site.
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Chymotrypsin active
site
Comfortably accommodates aromatics at S1 site Differs from other
mammalian serine proteases in specificity Diagram courtesy School
of Crystallography, Birkbeck College Biochemistry: Enzyme
Mechanisms 19 Feb 2008 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 Biochemistry: Enzyme Mechanisms 19 Feb 2008 iClicker quiz!
Why would the nonproductive hexokinase reaction H2O + ATP -> ADP
+ Pi be considered nonproductive? (a) Because it needlessly soaks
up water (b) Because the enzyme undergoes a wasteful conformational
change (c) Because the energy in the high-energy phosphate bond is
unavailable for other purposes (d) Because ADP is poisonous (e)
None of the above Biochemistry: Enzyme Mechanisms 19 Feb 2008
iClicker quiz, question 2: 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 Biochemistry: Enzyme Mechanisms 19
Feb 2008 Question 3: what would bind tightest in the TIM active
site?
(a) DHAP (substrate) (b) D-glyceraldehyde (product) (c)
2-phosphoglycolate (Transition-state analog) (d) They would all
bind equally well Biochemistry: Enzyme Mechanisms 19 Feb 2008
Convergent evolution Reappearance of ser-his-asp triad in unrelated
settings Subtilisin: externals very different from mammalian serine
proteases; triad same Biochemistry: Enzyme Mechanisms 19 Feb 2008
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.
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Cysteinyl proteases
Ancestrally related to ser proteases?
Cathepsins, caspases, papain Contrasts: Cys SH is more basic than
ser OH Residue is less hydrophilic S- is a weaker nucleophile than
O- Diagram courtesy of Mariusz Jaskolski, U. Poznan Biochemistry:
Enzyme Mechanisms 19 Feb 2008 Papain active site Diagram courtesy
Martin Harrison, Manchester University Biochemistry: Enzyme
Mechanisms 19 Feb 2008 Comments on the midterm
One remote student still hasnt taken it One local student still
hasnt taken it I have graded A-N plus a couple of Ps You may pick
up the exams beginning Thursday It was long, but many students
finished I dont do grade cutoffs, but if I did, I would guess that
theyre at 150 and 110. Rebecca Howard, Ph.D. Biochemistry: Enzyme
Mechanisms 19 Feb 2008