144

PII: S0307- 4412 (97)00070-8 Table 1 Initial velocity data obtained from the effect of various
concentrations of alanine on the enzyme glutamine synthetase at
Enzyme Kinetics: Partial and Complete different levels of glutamate
Competitive Inhibition Initial Velocity (Izmol dm 3 min 1)
CHRIS G WHITELEY [Ala] pmol dm 3/ 0.0 1.0 2.0 3.0 4.0 5.0
Department of Biochemistry and Microbiology
[Glu] (llrnol dm 3)
Rhodes University 2.0 0.242 0.213 0.195 0.182 0.172 0.165
Grahamstown 6140 2.5 0.271 0.242 0.223 0.209 0.199 0.191
South Africa 3.33 0.308 0.279 0.260 0.246 0.235 0.227
5.0 0.356 0.330 0.312 0.298 0.288 0.279
Introduction
The inhibition of enzyme activity is one of the major
ingly, this method has not met with overwhelming support
regulatory devices of living cells and one of the most
in undergraduate and/or postgraduate teaching areas. In
important diagnostic procedures of the enzymologist.
this department s it has been found that this type of
Such studies indicate specificity of an enzyme, the physical
and chemical architecture of the active site and the kinds approach, is an excellent replacement for the more usual
of enzyme-substrate and enzyme product complexes. Michaelis-Menten and/or Lineweaver Burk methods.
To illustrate Yoshino's method 7 in teaching enzyme
Enzyme inhibition is divided into two types: complete
and partial inhibition, otherwise known as linear and kinetics and to point out its advantages over conventional
procedures, a general rate equation is derived and specific
hyperbolic inhibition because graphs of reciprocal velocity
versus inhibitor concentration give a straight line and a kinetic interpretations are presented for competitive
hyperbola respectively? With complete enzyme inhibi- inhibitors.
tion, the velocity tends to zero when the concentration of From Scheme I it is possible to derive a generalised
the inhibitor increases while with partial inhibition the equation to relate [v/(Vo-v)] to the parameters in the
enzyme is converted into a modified, but still functional, scheme. Note: Vo is the velocity attained by the system in
enzyme-substrate-inhibitor (EIS) complex [Scheme I]. the presence of a fixed amount of substrate and no
inhibitor.
It follows that
E+$~ • E$ ~E+P

+ + K~ = [E][S]/[ES] or [ES] = [E][S]/Km (1)

I I
and
K~ = [E][I]/[EI] or [El] = [E][II/K~ (2)
K.' ¢k,
EI+$ ~ ~ EIS ~ E + I + P
and
Scheme I
K / = [ES][I]/[EIS l or [EIS] = [ES][I]/K,' (3)
Km and Kin' are the respective Michaelis constants for
substrate binding to enzyme and enzyme-inhibitor; I~ and
Ki' are the corresponding inhibitor constants and fl is the 13"
partiality factor. Although some graphical methods
including Dixon 2 and Cornish-Bowden3 are used for
kinetic analysis of the inhibitor action, these methods are
applicable to complete or linear inhibition only and not to
partial inhibition. The latter is usually analysed by the
replot of the primary reciprocal plot. 4 The Lineweaver
and Burk double reciprocal plot 5 provides the worst ~ 6.5-
estimation of the kinetic constants because of the
crowding of high-substrate concentration data points
close to the ordinate axis. Slope and intercept replots
cause a further magnification of errors in determining
kinetic parameters. Even the specific velocity plot 6 for
partial inhibition also requires secondary plots for the
determination of the kinetic parameters. -o.2 o 022 0:4 o16 o2a
Yoshimo 7 reported a graphical plot of fractional (dm-3/pmol)
velocity, [v/(Vo - v)] versus reciprocal of inhibitor concen- Figure 1 Fractional velocity plots showing partial competitive
tration (1/I). A simple inspection can distinguish whether inhibition of glutamine synthetase by alanine at different
the inhibition is complete or partial and whether it is concentrations of glutamate [2.0 pert (I), 2.5 ~ (&), 3.33
competitive, non-competitive or uncompetitive. Surpris- ([]) and 5 ~ (x)]

BIOCHEMICAL EDUCATION 25(3) 1997

 145

12- v ([E] [S]/Km) + (filES] [I]/K,')

/ Vo-v [E]+([E][I]/Ki)+(1--fl)([ES][I]/K/) (8b)
Ki = 3.6 I.tmol/dm-3
v ([S]/Km)+(fl[S][I]/Km .K/)
8-
Vo-v I+([I]/K~)+(1-fl)([S][I]/K,,.K/)

Simple graphical methods can be used to determine the

type of inhibition and the kinetic parameters of an
enzyme-catalysed reaction.
In competitive inhibition, the substrate binds to free
enzyme with a greater affinity than to the EI complex and
~ ' = ~ . With partial inhibition fl= 1 hence eqn 8
collapses to
-3 -1 i 3 5
S (~nol/dm-3) v ([S]/Km) ([S][I]/Km.K/)
Figure 2 A secondary plot of the slopes obtained from the lines V,,-v 1+ (I/K/) 1+ (I/K~)
from Fig 1 versus glutamate concentration
However, ([S]/Km)/(I + [I/K,]) can be rearranged to
so from eqns 1 and 3 K, (1 + [S]/Km)(1/[I]) (9)
[EIS] = [E][S][I]/K~.K/ (4) So
Now the initial velocity v/(V,, - v) = K, (1 + [S]/Km)(l/[I]) +K, (1+ [S]/K,.)IK,' (10)
= k3[ES ] +fl .k3[EIS] (5) For complete inhibition fi = 0, hence eqn 8 becomes:
and v/(Vo -- v) = K~(1 + [S]/Km)(1/[I ]) (11)

Vo = / 3 [ E T ] (6) Thus a graph of [v/(Vo-v)] versus the reciprocal of the

inhibitor concentration gives straight lines that converge
and on the abscissa at a point away from the origin (for a
ET = [E] + [ES] + [EI] + [EIS] (7) partial competitive inhibitor) and at the origin (for
complete competitive inhibition). Replots of slope and
Substituting and rearranging gives ordinate intercept of these primary plots are useful in
confirming the mechanism of inhibition and in the calcu-
v [ES] +fl[EIS] lations of the kinetic parameters.
(8a)
~-v [E l + [EI] + EIS[1 - fi]
Sample problem
From eqns 1, 2 and 4 Glutamine synthetase from E coli catalyses the amidation
of glutamate to produce glutamine. It is regulated by
inhibitors such as alanine.
1.5 Glutamine Synthetase
K.i = 3.6 gmoydm-3 I
/ L-Glutamic Acid+NH3+ATP r-~'~ L-Glutamine
Ki' =_8.33 ~znol/dm-3 Initial velocity data were collected by monitoring the
1 Km= 2.33 gmol/dm-3 uptake of ammonia in the presence and absence of
different concentrations of alanine (Table 1). Analyse
/ - these data, to determine the 'usual' kinetic parameters
and determine the mechanism of inhibition.

Solution 9,1°
From eqn 10 and the data (Table 1) fractional velocities
[v/(Vo- v)] can be calculated at the different substrate [S]
and inhibitor [I] concentrations. From this representation
(Fig 1) a series of lines, having slopes equal to
Kj (1 + [S]/Km), are seen which converge on the abscissa at
S (gmol/dm-3) -1//(,.' and intercept the ordinate axis at Ki(I+[S]/K,O/
Figure 3 A secondary plot of the intercepts on they axis of Fig 1 K/. This reflects a partial competitive inhibition
versus glutamate concentration mechanism.

B I O C H E M I C A L E D U C A T I O N 25(3) 1997
146
The parameters Kin, K~and K,' may be determined from
the two secondary plots of slope and intercept versus
substrate concentration (Fig 2 and Fig 31°).
Hence, Km= 2.33/~mol dm 3, K, = 3.6/~mol dm -3 and
Ki' = 8.33/~mol dm 3.
The parameter, Vmaxmay be calculated from simple
substitution into the Michaelis-Menten equation:
v = (V~,~[S])/(Km+ [S]). Hence Vm~x= 0.52/tmol
dm -3 min -1.
Equation 8 can also be suitably rearranged to allow
analysis of partial and complete non-competitive inhibi-
tion and partial and complete uncompetitive inhibition.
The author will be happy to supply the derivations and
suitable example problems to any interested reader. 9
References
1 SegelI, Enzyme Kinetics, pp 100-226. John Wiley,New York, 1973.
2 DixonM (1953)BiochemJ 55, 170-171
3 Cornish-BowdenA (1974)Biochem J 137, 143-144
4 ClelandW (1970)Enzymes, third edition 2, 1-65
5 LineweaverH and Burk D (1934)JAm Chem Soc 56, 658-666
6 Baici A (1981)EurJBiochem 119, 9-14
7 YoshinoM (1987)BiochemJ 248, 815-820
8 Department of Biochemistryand Microbiology,Rhodes University,
Grahamstown, South Africa (http://www.ru.ac.za/departments/
biochem/cw/html)
9 Suitable rearrangements of eqn 8 and sample problems may be
obtained fromthe author. (e-mail:chcw@giraffe.ru.ac.za)
10 Any suitable computer graphics package may be used. The authors
used Quattro.Pro (Borland.International)
PII: S0307- 4412(97)00094-0
Integrated P r o b l e m B a s e d L e a r n i n g for First Year
M e d i c a l Students: Does it Teach B i o c h e m i c a l
Principles?
CHARLES L HARRIS, a GUL GUNER,*
JAMES ARBOGAST, b LISA SALATI, 8
JAMES M SHUMWAY, c JOHN CONNORS d
and DIANA BEATTIE a
Departments o f Biochemistry, a Family Medicine b
Medicine and Pediatrics, Cand Physiology d
West Virginia University School o f Medicine
Robert C Byrd Health Sciences Center
Morgantown W V 2 6 5 0 6
USA
Introduction
The first year curriculum at West Virginia University,
School of Medicine (WVU SOM), was recently altered to
include an integrated problem-based learning (PBL)
experience? The purpose of these PBL sessions was to
equip first year medical students with skills in self-directed
learning and problem solving. A group of eight students is
led by a facilitator and given clinical cases fashioned in the
*Department of Biochemistry, Dokuz E'ylill University School of
Medicine, Inriralti 35340, Turkey.
BIOCHEMICAL EDUCATION 25(3) 1997
model developed at the University of New Mexico,2 but
written by clinical and basic science faculty at WVU. The
stated objectives of the integrated PBL course are to:
(1) integrate information across the basic science
disciplines,
(2) demonstrate the relevance of basic sciences in clinical
issues,
(3) enhance the retention of basic science knowledge,
(4) increase the student's ability to acquire knowledge
independently and to apply basic science concepts to
clinical cases,
(5) develop the student's communication and interper-
sonal skills in a group setting, and
(6) increase the level of intrinsic interest in the subject
matter, creating the motivation for learning.
In this article we describe the integrated PBL experi-
ence at WVU SOM, and assess the effectiveness of the
PBL process in identifying basic science issues relevant to
biochemical principles. The case discussed here is a
patient with non-insulin-dependent diabetes mellitus and
will serve to illustrate the approach. Since PBL groups
were led by basic science faculty who were not necessarily
experts in this area, we also assessed the uniformity of the
identified learning issues.
Background
The WVU SOM was established in 1902 with a two year
curriculum in affiliation with the College of Physicians
and Surgeons in Baltimore, MD. The School moved to its
current location in 1957 and inaugurated its four year
program in 1960 with the opening of a 550 bed University
Hospital. The Charleston Area Medical Center became
affiliated with the WVU SOM in 1972 as a branch campus
and offers third and fourth year clerkships for approxi-
mately one-third of the class. In the last few years, there
has been increased emphasis on primary and rural health
care at this institution with the establishment of additional
health care and educational centers in rural areas of West
Virginia. The WVU SOM admits approximately 88 new
medical students each year.
The curriculum is composed of two years of course
work in the basic sciences and two years of clinical clerk-
ships. The first year curriculum includes traditional
courses in gross anatomy, biochemistry, physiology,
neuroanatomy, behavior medicine, community medicine
and histology. In order better to integrate these courses
the PBL approach was implemented, with the intent that
exposure to clinical cases and the active learning process
involved in PBL would better prepare students for clinical
experiences in years three and four. Time spent in lectures
was reduced to make room for PBL. The present curri-
culum is currently undergoing revision, to an integrated
module approach, and will include earlier patient contact
and self-directed learning methods such as PBL.