Enzymology
59-464
Term Test I
Name : _________________________________
Student # ___________________
Signature: __________________________________
Instructions:
All answers are to be written in ink
By signing your name above you are agreeing to to the Departmental rules concerning Cheating
and Examinations found on the back of this examination
1) a) (3 pts) State the applications of Hammond’s Postulate that strengthen the Transition State Theory.
b) (5 pts) Discuss how the catalytic antibody approach illustrates the Transition State Theory and
Hammond’s Postulate.
a)
Hammond’s Postulate estimates TS analogue structures based on the assumption that unstable
intermediates and TS are similar in structure when similar in energy.
Structure elucidation of the transition state can lead to the design of transition state analogues
that can be used as enzyme inhibitors, and/or used to determine mechanism of enzyme reaction.
b)
- 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)
- The analog whose corresponding antibody contains the highest catalytic activity is then
predicted to have a structure that resembles the transition state, thus providing mechanistic
detail of the enzyme mediated reaction.
2) a) (3 pts) Define in terms of multifunctionality, the importance of metal ion catalysis in enzyme
systems.
b) (4 pts) Give a detailed example of an enzyme mechanism discussed in class that requires a metal
ion for catalysis.
a) - Electrostatic catalysis
- interaction with side groups to increase reactivity of enzyme
- neutralize cofactors for active site localization
- covalent catalysis
b) see lecture 4 slide 26
3) a) (1pt) What does PLP stand for?
b) (3 pts) What is the central feature that characterizes PLP mechanisms?
c) (3 pts) PLP is involved in different kinds of transformation reactions. How is reaction specificity
achieved?
a) pyridoxal 5’ phosphate
b) formation of Schiff base between enzyme then substrate (amino acid to be transformed)
also what is key is the resonance stabilization of carbanions that is provided by the
pyridine ring nitrogen (electron sink)
c) Substituent coordination
4) (4 pts) Briefly explain the advantage of current continuous flow techniques over stopped flow
techniques. What is the disadvantage?
Advantages - lower dead time possible due to faster mixing apparatus
- ultra early kinetic information possible
Disadvantages – can capture just one age at a time, larger amounts of reagents required
5) (5 pts) Compare the following two equations, (1) and (2). Which one correctly reflects the steady
state of the enzyme catalyzed reaction. Please justify your answer.
k1
E+S
k-1
ES
k2
k1[E][S]=k-1[ES] + k2[ES] ------------- (1)
k1[ES]=k-1[E][S]+ k2[ES] ------------- (2)
1) d[ES]/dt = 0
vformation = vdecomposition under steady state
E+ P
6) A scientist recently discovered a novel enzyme whose primary sequence resembles a family of
enzymes that commonly use a nucleophilic Cys residue and a catalytic acid Asp residue during
catalysis.
a) (6 pts) Using pH dependency experiments, steady state conditions, and a spectrophotometer,
describe the entire experimental setup that would allow this scientist to determine if indeed this enzyme
utilizes a nucleophile and a catalytic acid during catalysis. NOTE: the product of the reaction is
chromogenic with a known molar absorptivity value.
b) (3pts) What pH profile plot would best illustrate that the efficiency of this enzyme-mediated
reaction depends on a nucleophile and catalytic acid?
a) - check stability of enzyme at various pH
- use high amounts of substrate (saturating conditions) or determine Km, Vmax using
various [S]
- perform reaction versus time to obtain abs vs time plot
- slope of the line
- calculate initial velocity (vo) using Beer’s law
- perform the same reaction over stable pH range to see effect of pH on rate
- expect bell shape curve; rate increases at beginning due to [ ] increase in unprotonated
nucleophile (Cys), then rate decreases due to [ ] decrease of protonated catalytic acid
(Asp)
b)
left side shows ionization effect of nucleophilic Cys
right side shown ionization effect of catalytic acid Asp
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
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Programmable calculators and calculators with infra-red transmission capability are not permitted.
Absence
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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.
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Midterm examination
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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
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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)