Case Study - Would just like some jumping off points for the questions.... (Oct/06/2009 )

I haven't had any biology since high school so I'm really struggling with this because it's mostly biology.

The enzyme creatine kinase is important in energy metabolism and catalyzes the
reaction shown in Figure 10.1. The reaction is readily reversible in vitro. Enzyme
activity can be assayed by measuring hydrogen ion release (as determined by pH) in the
forward reaction or hydrogen ion consumption in the reverse reaction.
In order to understand a particular enzyme’s mechanism, scientists rely on a variety of
techniques. One preferred technique is x‐ray crystallography, which gives a threedimensional
picture of what the protein molecule looks like. X‐ray crystallographic data
are not always available however, since it is difficult to prepare the protein crystals that
are used in carrying out the analysis. In the absence of crystallographic data, scientists
use other experimental methods to determine the important features of an enzyme’s
catalytic mechanism. One technique is chemical modification in which various reagents
that might derivatize specific amino acid side chains are added to an enzyme solution.
Then the experimenter measures the activity of the enzyme using a specific assay, and
uses sequencing techniques to determine which amino acid has been modified. If a
chemical modification results in inactivation of the protein, it can be inferred that that
amino acid is essential for the enzyme’s activity in some way.
Another technique which is especially powerful is site‐directed mutagenesis in which
genetic engineering techniques are used to create mutant proteins with a single amino
acid substitution. In this manner, the role of specific amino acids in the catalytic
mechanism can be ascertained.


Sulfhydryl groups have the ability to react with the alkylating reagent Nethylmaleimide
(NEM) in chemical modification experiments (Figure 10.2). When
NEM is added to a purified solution of creatine kinase, Cys 278 is alkylated, but no
other Cys residues in the protein are modified. What can you infer about the Cys 278 residue based on this observation?


Based on the result described in Question 1, the investigators used the technique of
site‐directed mutagenesis to synthesize five mutant creatine kinase proteins in
which the Cys 278 was replaced with either a Gly, Ser, Ala, Asn or Asp residue. These
mutants were termed C278G, C278S, C278A, C278N and C278D, respectively, to
indicate the exact position of the amino acid change. The activities of the mutant
enzymes were measured in the presence and absence of specific cofactors.
a. All of the mutants had decreased creatine kinase activity as compared to the
wild‐type enzyme. What information does this give you about the wild‐type
enzyme mechanism?
b. The activity of the mutant enzymes C278D and C278N were compared and it
was found that the activity of the C278D mutant was 12‐fold greater than the
activity of the C278N mutant (although both mutants had lower enzyme
activities as compared to the wild‐type). Suggest an explanation for this
observation.
c. The activities of the mutant enzymes (although decreased from the wildtype)
were enhanced when either chloride or bromide ions were added to
the assay mixture. (An exception was the C278D mutant). Why do you think
that the ions were able to enhance enzyme activity?
d. The C278D mutant was an exception to the observation described in
Question 2c above. This mutant did not show an enhancement of enzyme
activity in the presence of chloride and bromide ions; in fact its minimal
enzyme activity decreased somewhat in the presence of these ions. Explain
why.


The results of the study presented here provided greater understanding for
previous chemical modification experiments that had been carried out with creatine
kinase. These reactions are shown in Figure 15.3. Some investigators modified the
Cys 278 with iodoacetamide and found that the enzyme activity was abolished as a
result. They concluded that the Cys 278 residue was absolutely essential for
enzymatic activity. But other investigators modified the Cys 278 with iodoacetate
and found that activity was decreased but not abolished, leading them to conclude
that the Cys 278 was not essential. Taking these results together with the results of
the current study, can you suggest an explanation that will clear up the confusion?


The investigators next carried out kinetic studies in which they measured the ability
of a second substrate to bind once the first substrate had bound. The data are shown
in Table 15.1. Kd refers to the constant for the binding of substrate to free enzyme
and KM refers to the constant for binding of that same substrate to the enzyme when
the other substrate is already bound to the enzyme. The results for the wild type
and two of the mutants are shown in Table 15.1.
a. Compare the Kd and KM values for creatine and ATP for the wild‐type enzyme.
What does a comparison of the Kd and KM values tell you about the ability of
each substrate to bind to the enzyme alone? to the enzyme when the other
substrate is present?
b. Similarly, compare the Kd and KM values for the two mutant enzymes. Again,
what does a comparison of the Kd and KM values tell you about the ability of
each substrate to bind to the enzyme alone? to the enzyme when the other
substrate is present?
c. Consider your answers to 4a and 4b and consider the Vmax values for both the
wild type and mutant enzymes. Assess the role of Cys 278 in the binding of
creatine and ATP substrates to the creatine kinase enzyme.

what are you trying to ask here? I don't get it.

The first part is a case study which i mostly understand the creatine kinase but the bottom half is questions and i'm not sure i really understand them all

These are straightforward questions and I imagine this should be placed in the "homework help" forum.
Question 1 and 2a can be answered easily from information gleaned from the first paragraph even without a knowledge of biology. For the remaining questions (2bcd and 3), I recommend that if you don't know the charge properties of the amino acids, you should examine a table of structures for the Gly, Ser, Ala, Asn and Asp residues and compare the properties of their side chains. Question 4 can be answered by examining Table 15.1 (not provided by you) and by reading the paragraph describing what kd and km are. If it is unclear to you, look up definitions of kd and km from other sources.
Hope this helps, but most people here won't just do your homework for you.
Good luck.