NAGGING IS MY HUMBLE LITTLE FORTE


A couple of months back, I was "surfing" around IVLE - an online "blackboard" for students (which is not unlike OLE used in Temasek Polytechnic) to receive updates/announcements and to download their lecture notes and tutorials and assignments and all the irritating rubbish - when something interesting, and kind of hilarious, caught my pretty, dainty, sparkling, gorgeous, and much-coveted pair of mesmerizing eyes.






This is the main IVLE webpage.








And this is a particular section of the Gradebook - an online facility which has, thus far, never been used by any of my Chemistry lecturers; save for a certain Biochemistry lecturer maverick.










A zoom-in of the words....





Still cannot decipher them?

Well then, let the lovely and benevolent Princess alleviate your agony. The first line actually reads: Q1 + Q3: Information overkill?!






Needless to say, I was dumbfounded. Since that particular assignment was uploaded for submission some donkey-monkey-cronky months ago, I retrieved that particular file from amongst the assignments I've saved in my ever-reliable lappy.



Q1: Consider the following reaction which is catalysed by a group of similar enzymes. Which Enzyme Commission class should the group of enzymes be classified under? Explain how you had arrived at your answer. (2 marks)

MY ANSWER:
Under the Enzyme Commission (EC) Classification of enzymes, this particular enzyme should be classified under Class 3 – Hydrolases. More specifically, this enzyme should be classified under EC 3.2. – Glycosidases (also known as glycosylases). Under EC 3.2.1. these enzymes hydrolyse O- or S-glycosyl compounds.

Hydrolases are enzymes which cause hydrolysis reactions – that is, cleavage of bonds by using water. The water molecule – H2O – is dissociated into a proton (H+) and a hydroxide ion (OH-). Upon bond cleavage - which, in this case, produces monosaccbaride products – H+ or OH- are added into the C1 one of the two sugar molecules involved in the bond.

Under the EC, there are a total of 6 different classes of enzymes.

1) Oxidoreductases – enzymes that catalyse a redox reaction. It involves the transfer of electrons usually in the form of hydride ions (H-) or hydrogen atoms.

2) Transferases – enzymes that transfer a function group from one molecule to another.

3) Hydrolases – enzymes that bring about cleavage of bonds by hydrolytic reactions.

4) Lyases – enzymes that break C-O, C-C, or C-N bonds.

5) Isomerases – enzymes that rearrange/transfer functional groups within a molecule to yield isomeric forms.

6) Ligases – enzymes that join two molecules; formation of C-C, C-S, C-O, and/or C-N bonds by condensation, coupled with ATP cleavage.



Q3: An enzyme belonging to this group of enzymes was isolated from a bacterial culture. It was then found to have a molecular mass of 266kDa. However, when the enzyme was denatured with urea and mercaptoethanol, two different values of 105 kDa and 150kDa were obtained. Explain the observed results. (3 marks)

MY ANSWER:
Most enzymes are not just merely a single peptide strand. Instead, they are usually a combination of several proteins – that is, they are an aggregate of smaller globular proteins. Thus, enzymes can also be referred to as quaternary proteins – that is, these proteins are made up of several subunits of tertiary-structured proteins. In addition, there might also be the presence of prosthetic groups (i.e. organic molecules such as vitamin, sugar, lipid) or inorganic molecules (e.g. metal ions such as Mg2+) incorporated into the quaternary structure of the protein. Such proteins – with prosthetic groups – are called holoproteins or conjugated proteins. These prosthetic groups usually bind to the peptide chain via covalent bonds.

Urea acts by breaking up hydrogen bonds, while mercaptoethanol breaks disulphide bonds. When treated with urea and mercaptoethanol, only the Hydrogen bonds and disulphide bonds (both of which, are non-covalent bonds) are cleaved; while the covalent amino acid backbone (made up of covalent peptide bonds) of the primary structure of the protein is not affected.

When the enzyme, with a molecular mass of 266kDa, is treated with urea and mercaptoethanol, two different protein subunits are obtained – with one weighing 105kDa, and the other weighing 150kDa. The difference in molecular mass could be due to a number of reasons – such as the dissociation of prosthetic groups, or the running of SDS-PAGE etc.

Even though most prosthetic groups usually bind to the peptide chain via covalent bond, there is still a possibility of cleavage of certain non-covalently-bonded prosthetic groups (e.g. lipoproteins or their derivatives may be bound to proteins either covalently or non-covalently http://en.wikipedia.org/wiki/Lipoprotein). Thus, when the enzyme is treated with urea and mercaptoethanol – both of which affect non-covalent bonds – the non-covalently bonded prosthetic groups might get dissociated, and this results in a decrease in the overall molecular mass.

Alternatively, the discrepancy in molecular mass could be due to the protein separation technique – electrophoresis. For proteins, a discontinuous polyacrylamide gel is used as a supporting medium, while negatively-charged Sodium Dodecyl Sulphate (otherwise known as SDS) is used to denature the proteins. This particular technique involving the separation of protein chains is, more specifically, known as SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis).

SDS destroys the complex structure of proteins, reducing it to its primary structure (i.e. linear chains). Being negatively-charged in nature, the SDS-denatured proteins will be attracted to the anode when the polyacrylamide gel is run through an electrical discharge. Thus, the final separation of proteins will depend almost entirely on the difference in molecular mass in peptide chains – the bigger the peptide chain (i.e. the more the molecular mass), the slower it’ll migrate down the gel. The obtained protein bands can be tallied and matched with the protein molecular mass “ladder” standard.

For this case, the quaternary-structured enzyme – being more complex in structure – is naturally harder to denature than the protein subunits, which are tertiary in structure.

Thus, the quaternary-structured enzyme might not have been fully denatured yet when SDS-PAGE was carried out. This results in a higher molecular mass reading. In contrast, the tertiary-structure protein subunits are denatured to a linear state, and thus, are able to “travel” down the polyacrylamide gel more efficiently. Hence, this results in a lower – and more accurate – molecular mass reading.



Okie, fine, I admit it's a little too extreme for questions which are allocated only 2 marks and 3 marks respectively. But hey, this is generally what educators get when students (in particular such as yours truly) are given 1 whole entire week to complete an OPEN-ESSAY assignment - with no word limit - which is to be uploaded online into IVLE by (insert date), (insert time).
Saves them the hassle of printing and dropping the assignments into the "pigeon hole", you see.


After all, isn't there a saying which goes "Better late than never"? Similarly, it's better more than less, too (my cool logic =P).


Anyway, I think my tutor marked my assignment script until his eyes nearly combusted. Maybe that's why he resorted to adding an exclamation mark to the end of his sentence (Information overkill?!) to express his amusement and exasperation, comical effect not withstanding.


I think he's pretty nice already. If I were him, I would have gave a big, ugly X cross, and smack the student upside down and inside out.

So young, then so lor soh like some old granny like that. HOW CAN???!!!