Enzymes as Tools of Discovery

This was probably the most interesting lecture in the Enzyme Kinetics module (that we've had so far, that is- our last lecture's tomorrow). Let's see what it entails!

Ribozymes

Ribozymes are enzymes that are made out of RNA (rather than protein). They usually catalyse reactions involved in the synthesis and processing of other RNAs. For example ribozymes, helped along by guanosine, catalyse the removal of introns in the processing of Tetrahymena rRNA. (As far as I know we don't have to know the mechanism in detail, so let's not get bogged down in the details.)

Abzymes

Abzymes are basically enzymes that are designed to catalyse particular reactions. Abzyme design is based on two main principles: that enzymes catalyse reactions by binding the transition state very tightly, and that antibodies bind things very tightly. Hence, the idea here is to make antibodies against transition states.

Abzyme creation isn't too hard to understand: first you choose a molecule that is very similar to the transition state (you can't choose the transition state itself as it's very unstable) and then you raise antibodies against that transition state. How those antibodies are raised isn't something that we need to know for now, but it'll probably come up if you're going forth in Biochemistry.

Enzymes as Tools

Being able to design enzymes is all very well and good, but that probably wouldn't be so useful if we couldn't use enzymes as tools! Enzymes, both natural and designed, can be useful tools thanks to their specificity and their ability to catalyse a lot of reactions so that only a small amount of enzyme is needed.

Here are some examples of where enzymes can be used as tools:

• Restriction enzymes- used to cut DNA.
• Specific proteases- used to cut proteins.
• PCR- used to amplify DNA segments.
• ELISA (Enzyme-Linked ImmunoSorbent Assay)- uses a small amount of enzyme to catalyse the formation of a large amount of product, thereby allowing small amounts of antigen to be detected.
• Markers for cloning- see my earlier post to see how cloning and enzyme activity/inactivity can be used to see whether a plasmid has taken up a vector or not.

I'm not going to talk about restriction enzymes or PCR, as they've been done to death on this blog. I also won't go into any more detail about cloning markers, as this lecture didn't go beyond what I mentioned on that earlier post.

As for proteases, I won't go into much detail on the proteases themselves (the next post will be about proteases) but what I will talk about is a funky 2D gel on that slide that can be used to separate out proteins. The reason it's called a 2D gel is because proteins on it are separated out according to two characteristics: size and pKa. Along one side of the gel there is a pH gradient, and proteins will settle at the pH that matches their isoelectric point (i.e. when they're neutrally charged- see my earlier post for a bit more information about isoelectric points). Perpendicular to this gradient, a charge is run across the gel, just like in agarose gel electrophoresis. This separates proteins out according to their size.

After this is done, you can cut out a part of the gel containing the protein that you're interested in. From there, you can use proteases to cut the protein, and mass spectrometry to find the size of the fragments. You can then use this information to search databases to find out what protein you're looking at.

Now I'll talk a little bit about ELISA and give an example of how it's used to detect HIV!

The first step in testing for HIV is to make some rabbit antibodies against it. This involves giving rabbits some of the HIV coat proteins- not the whole virus, just the coat. The rabbits can then raise IgG against it.

Some more important molecules include horseradish peroxidase and goat IgG. Horseradish peroxidase can bind to goat IgG. You'll see why this is important in a bit.

Samples containing HIV can be put into a polystyrene dish, with rabbit IgG added. This is then washed to remove unbound antibodies. The horseradish peroxidase/goat IgG complex can then bind to rabbit IgG (yup, they can all bind together, how convenient). The horseradish peroxidase can then do its work in converting colourless substrates into coloured products (unfortunately, I don't know what substrates/products are involved in this reaction). The amount of coloured product formed is proportional to the amount of peroxidase present.