Enzymology
59-464
TERM I - 2005
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Instructions:
All answers are to be written in ink
By signing your name above you are agreeing to the Departmental rules concerning Cheating
and Examinations found on the back of this examination
1) (6pts)
(a) Is it an advantage for the active site of an enzyme to complement the structure of the reactant
(substrate) or the transition state? Explain why in terms of rate acceleration.
(b) Describe an experimental approach that provides evidence to your answer in part (a).
(a) Transition state
-TS is the highest free energy species on rxn coordinate due to unfavourable charges and
abnormal conformations, leading to high activation energy barrier and slow reaction rate
- Enzyme active sites stabilize TS by neutralizing unfavourable charges/conformations, using
binding energy to “pay” for decrease in entropy, and stabilizing transition state
- This leads to decreasing activation energy of reaction and increasing rate of reaction
(b) Catalytic antibodies
- produced a variety of analogues whose ground state geometry resembles structure of
substrate and predicted transition state(s)
- injected analogue immunogens into separate animals to produce antibodies
- tested antibodies for activity against the substrate in the absence of enzyme
- antibodies raised against transition state displayed rate enhancement compared to no enzyme
(although less than WT enzyme)
Other acceptable explanations
-lysozyme example (properly explained)
-transition state analogues as enzyme inhibitors (properly explained)
Compare TS analogues ot substrate analogues to show that TS analogues were better enzyme
inhibitors
2) (6pts) The removal of the metabolic product CO2 from tissues such as muscles is a crucial aspect
of respiration. Explain how the remarkably efficient carbonic anhydrase enzyme uses two molecules
of water and a Zinc ion to convert CO2 to HCO3 (bicarbonate). Include a complete structure of the
zinc coordination sphere of the active site (amino acid side chains, zinc ion, and water).
See Lecture 4 slides 24 and 25
3) a) (3pts) Define Km, Vmax, and kcat conceptually (i.e. without reference to algebra).
Km – substrate concentration when enzyme is operating at ½ Vmax. Measure of substrate
affinity
Vmax – maximum rate saturation kinetic conditions (when all enzyme active sites are saturated
with substrate – [ES] is in steady state
kcat - maximum number of substrate molecules converted to product per unit of time (the
turnover number, catalytic constant)
b) (3pts) How do these values change with increasing enzyme concentration?
Km does not change
Vmax increases
kcat does not change
4) (3pts) A novel enzyme was tested at a concentration of 10 μM for its ability to hydrolyze a spectrum
of potential substrates yielding the following kinetic parameters:
A
B
C
D
E
1.0
0.44
0.81
0.49
1.2
Vmax (μM min-1) 11.5
9.80
Km (mM)
13.2
11.7
10.4
Which substrate would be hydrolyzed most rapidly? Show Proof.
kcat = Vmax/ET
kcat/Km = efficiency constant – best indicator of substrate hydrolysis
Proof – units and correct kcat/Km numbers
Substrate D
5) (4pts) Explain how to determine the molar absorptivity (extinction coefficient) of an analyte
(product)
-
Find λmax by using one [S] and measuring absorbance vs wavelength
At λmax measure absorbance at various [S] concentration and plot. Should be linear
Slope of line is molar absorptivity (ε = L/mol cm)
6) (6pts)
(a) State three purposes of obtaining pre-steady state measurements for an enzyme mediated reaction.
(b) Name three analytical methods commonly used to obtain pre-steady state kinetics data.
(c) Briefly describe one of these methods.
(a) see lecture 9 slide 4
(b) see lecture 9
(c) see lecture 9
7) (2 pts) Circle the correct answer concerning enzyme inhibition
(a) Competitive inhibitors decreases the kcat of enzyme mediated reactions
(b) Uncompetitive inhibitors increase Km and decrease Vmax of enzyme mediated reactions
(c) The fraction of enzyme not available for catalysis is represented by the factor (1 + KI/[I])
(d) Noncompetitive inhibitors bind to the free enzyme or the enzyme-substrate complex
8) (2pts) Circle the incorrect answer concerning chemical catalysis by enzymes
(a) Ground State destabilization is an example of Electrostatic catalysis
(b) Enzymes require cofactors in order to perform Electrophilic catalysis
(c) The proximity affect of intermolecular catalysis achieves rate enhancement by desolvating the
substrate from water
(d) The bell-shaped curve of an enzyme activity with respect to pH is indicative of the
involvement of both a general acid and base in catalysis.
Department of Chemistry and Biochemistry Guidelines
for Examinations, Assignments and Laboratory Reports
Cheating and Plagiarism
1. During an examination, students must not have in their possession any unauthorized books, notes, or
extraneous material, unless permitted by the instructor.
2. All incidents of cheating and plagiarism will be reported by the instructor directly and immediately
to the Departmental Head for consideration of disciplinary action as delineated in Senate By-law 31.
Calculators
1. Students may only use calculators approved by the Faculty of Science and/or their instructors.
Programmable calculators and calculators with infra-red transmission capability are not permitted.
Absence
1. Attendance of laboratories and mid-term examinations is MANDATORY.
2. Students who miss a mid-term examination or laboratory must provide written documentation to
justify an absence. Unexcused absences or incomplete laboratory reports will result in a grade of
incomplete, which in turn will result in a grade of incomplete for the course.
3. Such a student should call the departmental office 253-3000 x3521 to report his/her name and the
examination missed within 24 hours of the exam.
4. Written documentation justifying the absence must be presented within 48 hours of the examination
or as soon as possible.
5. Excuses will not be accepted after a student has taken an examination.
Midterm examination
1. Only examinations written in non-erasable ink will be considered for a grade appeal.
2. All grade appeals must be accompanied with a written rationale for the grade appeal.
Requests such as "see question xx" contain insufficient information and will not be considered for a
grade appeal.
3. All grade appeals must be made either within five working days after the examination is returned to
the class, or by a date designated by the professor.
Laboratory Reports/Assignments
1. Plagiarism is defined in section 2.4.22 of the University Calendar. Students are reminded that
copying laboratory reports and assignments constitutes plagiarism. When two or more laboratory
reports/assignments containing substantially identical material are submitted, a grade of 0 will be
assigned to each student, and the incident will be reported to the Department Head.
Final Examinations
1. A student may inspect his/her own corrected final examination.
2. All posted final grades are unofficial, and non-negotiable.
3. Students who wish a formal appeal of their final examination/grade as described in section 2.6 of the
Calendar may complete the appropriate paperwork at the registrar's office. In general, successful
appeals will be based solely on academic merit. Grade appeals to satisfy admission or scholarship
requirements or other program prerequisites will be rejected.
(These guidelines were approved by the Departmental Council on 19 Dec. 1996; updated Sep. 2003)