BIOCHEM 353- Enzymology SyllabusDr. Scott Morrical [e-mail]

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BIOCHEM 353- Enzymology Syllabus Dr. Scott
Morrical smorrica_at_zoo.uvm.edu Dr. Steve
Everse severse_at_zoo.uvm.edu WF 130 245 PM,
C447 Given Dr. Chris Berger christopher.berger_at_u
vm.edu September 10 Intro Steady-state
kinetics Morrical 12 Steady-state
kinetics Morrical 17 Steady-state
kinetics Morrical 19 Steady-state
kinetics Morrical 24 Transient-state
kinetics Berger 26 Transient-state
kinetics Berger October 1 Transient-state
kinetics Berger 3 Transient-state
kinetics Berger 8 Exam 1 (kinetics) 10 Allos
terics Morrical 15 Allosterics Morrical 1
7 Allosterics student paper presentations 22
Allosterics student paper presentations 24
Allosterics student paper presentations 29
Allosterics student paper presentations 31 Ex
am 2 (allosterics) November 5 Mechanism
active site Everse 7 Mechanism active
site Everse 12 Mechanism active
site Everse 14 Mechanism active
site Everse 19 Mechanism active
site Everse 21 Mechanism active
site Everse 26 Mechanism active
site Everse 28 THANKSGIVING BREAK (no
class) December 3 Mechanism active
site Everse 5 Exam 3 (enzyme
mechanisms) Classes will be a mixture of
lectures, computer labs, and student
presentations depending on instructor and topic.
Exams may be in-class or take-home format
depending on instructor and topic.
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On-Line References for Steady-State Enzyme
Kinetics Dr. Peter Birch, University of
Paisley http//www-biol.paisley.ac.uk/kinetics/con
tents.html University of Texas http//www.cm.utex
as.edu/academic/courses/Fall2001/CH369/LEC05/Lec5.
htm Terre Haute Medical College http//web.indsta
te.edu80/thcme/mwking/enzyme-kinetics.html
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Enzymes-- Biological Catalysts Catalyst- a
substance that increases the rate of a reaction
without itself being changed or consumed  in the
overall process Turnover- the catalyst may be
reused in subsequent reactions
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Chemical Kinetics Equilibria The position of
equilibrium is determined by the free energy
change, DGo DGo -RTlnKo The rate of a
reaction depends on the free energy of
activation, DG
Catalysts Speed up reactions by lowering the
free energy of activation, DG Do not affect the
position of equilibrium (DGo unchanged)
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• Why do we need enzymes?
• A chemical reaction occurs only if the
  molecules possess a minimum amount of
  energy---Activation Energy
• Chemical reactions require initial input of
  energy--usually in the form of increased heat
• Raising the temperature increases the rate of
  (vibrational, translational) movement of the
  molecules and the chance of collision
• An increase in the concentration of reactants
  can also increase the chances of a chemical
  reaction occurring
• HEAT and MORE REACTANTS can increase chance of
  chemical reaction occurring
• Biological systems cannot raise heat or
  concentrations at will

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How do enzymes do that? Provide alternate
pathway by lowering energy of activation,
stabilization of transition state--same as adding
heat Lowers activation energy, but does not
change free energy required for the reaction to
occur (alters the rate, but not
thermodynamics) Provide a surface for the
reaction to occur, bringing reactants into close
proximity to each other--functional equivalent of
increasing concentration
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Enzyme catalysts contain unique active
sites---where the substrates bind and the
reaction takes place Lock and key
model--substrate fits exactly into active site
Induced fit model--substrate causes change in
enzyme's active site shape to make substrate fit
Once bound, the substrate reaches the
transition state and bonds are rearranged. The
enzyme active site Places atoms in close
proximity to each other Orients substrate
correctly These two effects facilitate the
breaking and reforming of bonds
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Enzyme-Substrate Binding Specificity
Lock Key
Induced Fit
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Classes of enzymes Oxidoreductases--oxidation
/reduction requires a co-factor such as NAD or
FAD A B gt A B Transferases--transfer
of a functional group A-B C gt A B-C
Hydrolases--hydrolysis of functional group by
water A-B H20 gt A-H B-OH
Lyases--elimination to form double bond or
addition to a double bond X-A-B-Y gt AB
X-Y Isomerases--isometric interconversions X-
A-B-Y gt Y-A-B-X Ligases--ATP dependent
joining of two molecules A B ATP gt A-B ADP
Pi
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Enzymes Compilations of Databases Online
Resources
http//restools.sdsc.edu/biotools/biotools12.html
FRONTIERS IN BIOSCIENCES http//www.bioscience.o
rg/urllists/protdb.htm ExPASy Molecular Biology
Server http//us.expasy.org/
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Enzyme Kinetics Studies of the rate or velocity
of enzyme-catalyzed reactions, and factors
influencing these rates. Mathematical analyses
of the relationships between substrate (or
inhibitor, activator) concentrations and
reactions rates yield -- characteristic
properties of enzymes or classes of
enzymes -- insights into enzyme mechanisms and
physiology
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The Effects of Substrate Concentration on
Reaction Rate
S substrate P product E enzyme
E
S P
Uncatalyzed Reaction
At a fixed concentration of enzyme, the velocity
reachs a maximum, which fits the equation of a
rectangular hyperbolic curve y(ax)/(b x)
v
S
Velocity v dP/dt -dS/dt
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Steady-State Derivation of Michaelis-Menton
Equation (Briggs Haldane)
Upon mixing of enzyme and substrate ES rises
rapidly and reaches a steady state, where the
rate of formation and breakdown of ES are
equal, i.e.   v1 k1 ES and v2 k-1 ES
k2ES so that at steady state v1 v2 Free
enzyme conc. E Et -ES note that E
cannot be measured, but Et is known, as is S
since initial velocities, and P can be
measured. Now solve for the unknown ES. v1 k1
(Et - ES)S v2 k-1ES k2ES
rearrange (Et -ES)S/ES k-1 k2 /k1
Km Solving for ES gives ES EtS/ Km
S the velocity (v) of the reaction will be
proportional to the formation of ES, so v k2
ES (substitute in the value for ES in red
above) to get vk2 EtS/ Km S and
at saturating S, Vmax   k2Et (substitute
Vmax for the k2 Et in the above equation),
then   v Vmax S/ Km S which is the same
as the Michaelis-Menton equation
time
Note importance of initial velocities (vo)
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The Effects of Enzyme Concentration
Vmax is directly proportional to enzyme
concentration
At saturating substrate (S gtgt Km)
Km is independent of enzyme concentration
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Measuring Kinetic Parameters Graphical
Computational Methods Lineweaver-Burk
Plot Eadie-Hofstee Plot Hanes Plot
Direct Linear Plot Direct Fitting of v vs.
S Curve Single Progress Curve Statistics
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Lineweaver-Burk Plot Rearrangement of
Michaelis-Menten equation to linear form 1/v
(Km/V)(1/S) 1/V
Plot for hypothetical enzyme with V 10 Km
4
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Disadvantages of Lineweaver-Burk Plot
Still working with hypothetical enzyme with V
10, Km 4
1/v (Km/V)(1/S) 1/V
only here random error has been introduced into
multiple data sets, and results plotted
to illustrate how greatly estimates of Km and V
can vary from plot to plot depending on
data quality. How to improve --averaging --weigh
ting --choose another method
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Eadie-Hofstee Plot Rearrangement of
Michaelis-Menten equation to another linear
form v -Km(v/S) V
Same hypothetical enzyme with V 10 Km 4
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Disadvantages of Eadie-Hofstee Plot
Still working with hypothetical enzyme with V
10, Km 4
v -Km(v/S) V
Once again random error has been introduced to
demonstrate the scatter which can skew estimates
of Km and V almost as bad as Lineweaver-Burk
plot. Another problem Both axes are functions
of the dependent variable (v)
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Hanes Plot Rearrangement of Michaelis-Menten
equation to still another linear form S/v
(1/V)S Km/V
Same hypothetical enzyme with V 10 Km 4
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Error Issues in the Hanes Plot
Still working with hypothetical enzyme with V
10, Km 4
S/v (1/V)S Km/V
Once again random error has been introduced
generally scatter is improved relative to LH and
EH plots, which can improve the accuracy of Km
and Vestimates. Avoids the other problem of EH
plots in that dependent variable (v) does not
influence the idependent (horizontal) axis.
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Direct Linear Plot Here, using two data points
(w/o error) from same hypothetical enzyme with V
10 and Km 4 For both (S, v) data points,
plot -S on horizontal axis and v on vertical
axis, then draw a line connecting the two
values. The lines intersect at coordinates
(Km, V) allowing direct read-out of these
parameters.
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Direct Linear Plot Here is the same type of
plot only with lines drawn for all 10 of the
error-free (S, v) data points from our
hypothetical enzyme with V 10 and Km
4 Since there is no error, all of the lines
intersect at a common point with coordinates
(Km, V).
V
Km
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Effects of Error on Direct Linear Plot Now
looking at 5 (S, v) points from a data set
including random error, again from our
hypothetical enzyme with V 10 and Km
4. Lines no longer intersect at a common
point, thereby giving a large range of values for
Km and V from all of the different intersection
points. In fact, the number of intersections is
given by the simple equation n(n-1)/2, where
n the number of lines. Statistically, the
best way to deal with this is to take the median
(not the mean) values of Km and V, which
minimizes the contributions of spurious outliers
(such outliers could badly skew the mean values).
See Birchs website for a good description of
this approach.
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Theory Behind Direct Linear Plot Yet another
linear rearrangement of the Michaelis-Menten
equation V (v/S)Km v would give a
straight line if constants V and Km plotted
against each other slope and intercept would be
variables v/S and v, respectively. In
fact, lines in Direct Linear Plot represent
infinite number of values of Km and V which
satisfy the Michaelis-Menten equation for a given
(S, v) data pair. But the intersection of
two lines generated from two different (S, v)
data pairs gives the unique values of Km and V
that satisfy both conditions, so these must be
the true ones (subject to experimental error of
course).
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Direct Fitting of v vs. S Curves A number of
commercially available non-linear regression
packages exist which will perform global fits of
the hyperbolic form of the Michaelis-Menten
equation to experimental data. -- Enzfit,
Kaleidograph, etc. -- Outputs usually consist of
Km and V parameters with statistical arguments
such as variance and other indicators of
goodness-of-fit. -- CAUTION Always compare
fitting results with standard graphical methods
of analysis. Be aware that different statistical
packages can skew Km and V values depending on
how data is weighted and how outliers are
treated. U. Paisley Website Includes
Instructions For Setting Up a Global Fitting
Routine for Michaelis-Menten Enzyme Kinetics Data
in Microsoft Excel http//www-biol.paisley.ac.uk/k
inetics/Chapter_2/contents-chap2.html
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Km and V From a Single Progress Curve
Substrate concentration is monitored throughout a
reaction timecourse At any given time
(substrate conc.), the instantaneous velocity is
determined from the slope of the curves tangent
line. (S, v) data pairs so generated are
analyzed via any of the methods already discussed.
Pitfalls (there are many) -- not based on
initial velocities. -- product inhibition. --
noise affects slope. -- enzyme and/or substrate
lability. -- more susceptible to changes in pH,
ions, etc.
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Siesta Time!