Welcome.
We are discussing biomolecules and their relationship to cell structure and function.
We have our own stories through which we pick up these things; we have had a few stories
so far.
If you, the main messages from the story so far has been that there is there are microorganisms
and they (are) they can be categorised into various different domains.
The major biomolecules, I keep mentioning this although I haven’t told you everything
so far, the major biomolecules are four, we first saw lipids which are water insoluble
substances that are soluble in organic solvents, reasonably vague definition but that’s what
it is, we saw some examples of it.
And then we also said that lipids form an important component of the membrane, of the
cell membrane, cell membrane is an important cell envelope; some cells have only a cell
membrane to distinguish themselves from the environment, some cells have a cell membrane
and on top of that a cell wall too, and maybe there are other layers, that we’re not talking
about now, (we’re) let’s not get into that.
The nature of the lipid itself makes it possible for lipids to act as or lipid bilayers to
act as effective envelopes between the cell and their surroundings.
I probably briefly mentioned that in the passing earlier, let me talk a little bit about that
and then go further.
The, if you look at the way the lipids have organised themselves in the membrane there
are, let’s go here it might be a better picture; there are lipid bilayers.
This is water maybe extracellular.
This is water intracellular, right?
And this membrane in three-dimensions, separates the intracellular the extracellular.
If you look at the orientation of the lipids here, the hydrophilic heads are oriented towards
water naturally because the hydrophilic means water loving and they would like to be oriented
towards water.
And hydrophobic, like likes like, therefore they orient towards each other and by their
very nature there is a hydrophilic layer here, there is a hydrophobic core here, and a hydrophilic
layer here, separating the intracellular from the extracellular parts.
And this bilayer can self-assemble, nobody needs to put in any energy to assemble these
layers by the very nature of their molecules, here is water, here is water and therefore
the water loving parts will orient towards water and the water hating parts, the hydrophobic
parts, this is hydrophobic here, hydrophobic part will come together here.
If we think about it, this forms an effective barrier to the cell from its surroundings.
Substances cannot very easily pass through the membrane; molecules cannot very easily
pass through the membrane because of the nature of the membrane.
Here you have water loving-water loving so that’s okay.
But when water loving molecule comes here it needs to pass through a water hating core
and the rates of pass through the membrane would be slowed down significantly.
In a typical cell, there are actually proteins, you know the, cell membrane is fluid mosaic
of these lipids and proteins, some of those proteins actually function to transfer substances
from the outside to the inside, glucose goes from outside the cell to the inside of the
cell, through some such proteins and many other things have transporters that make it
possible for such substances to move from the outside of the cell to the inside of the
cell at reasonable rates.
So by the very nature of these molecules they act as effective barriers to outside components
and they protect the cell.
So this is one of the important structure function relationships compared to lipid.
We have already seen that proteins, amino acids, polymers of amino acids are proteins
and they are a large class of important biomolecules in the cell and there is a structure function
relationship, one of which we have seen.
By the very nature of amino acids and the polymerisation of amino acids into proteins,
different parts of the proteinaceous chain would attract depending on the side groups
that are there and their interactions with water, all these combined, there could be
interactions between molecules that are on different parts of the chain.
Because of that the protein molecule folds, and because of the proper folding the protein
molecule is able to act as an enzyme or even carry out its other functions, non-enzymatic
functions and so on.
So by the very nature of the polymers of amino acids they get their function.
And if the appropriate folding of the haemoglobin chain leads to the haemoglobin protein when
it folds properly, then it is able to carry oxygen and the red blood cells looks nice
and clean like this disc shaped structure, whereas if in the sixth position the glutamic
acid gets changed to valine for some reason, then the structure entirely goes out of proper
orientation and it is no longer able to carry oxygen properly, not just that it makes the
shape of the red blood cells sickle shaped, and we get (sickle cell) sickle cell anaemia
because of this.
So there is a good structure function relationship between these molecules.
We have so far seen lipids, carbohydrates, carbohydrates as you know are intermediates
in metabolism, they are a large energy stores and so on so forth.
We will look at that in in some detail later in the lectures.
And then of course proteins which (are) which form very many different functions in the
cell because of the nature of the structure.
Having said these I think in the last class we stopped here the structure function relationship.
Let’s take things further from here in this lecture.
Let us, okay, we will take a side story now, okay?
We are still in the major story of curd making, we are asking different questions and trying
to answer them.
As a part of answering them, we are uncovering very fundamental aspects of biology, biological
molecules and so on.
Now our side story here is glycolysis again, you know, we know that glucose going to pyruvate
through glucose 6-phosphate, fructose 6-phosphate and so on so forth, is called glycolysis,
these set of reactions and the curd formation happened because of the acid, the lactic acid
that is produced from pyruvate through a couple of steps, gets released into the medium and
the pH (we) we will get to that (and) and this causes curd formation, right?
If we look at this glycolysis, focus on glycolysis, all these are carbohydrates, fine.
However, the conversion from one carbohydrate to another, glucose to glucose 6-phosphate,
glucose 6-phosphate to fructose 6-phosphate and so on so forth.
Each step is, of this so called metabolic pathway, glycolysis, is catalysed by enzymes,
okay?
Each of these steps is made possible at the temperature of the cell because of a catalyst
called enzymes, and all such enzymes that we’re concerned with, are proteins.
There are other molecules in the cell that have enzymatic activities such as RNA, we
will keep that aside for the time being, we’ll just focus on the majority of enzymes in the
cell in terms of glycolysis, in terms of metabolic pathways and they are all proteins.
Proteins play huge roles in every cell and they make the various functions of the human
body possible, okay?
I would like you to watch this particular video if you look at the number here, it is
number 14, role of enzymes in the human body.
It’s a nice short, let’s say 3 to 4 minute video, I think.
If you watch that you will get an idea as to the variety of functions that enzymes perform
in the human cell and different human cells put together as the human body, and the, why
humans do what they do, the biochemical basis is all given, is all made possible because
of the enzymes, biochemical basis is the enzymes.
So please take a look at this video.
Let us talk a little bit about these enzymes which are specialised proteins or a class
of proteins.
Enzymes can be highly specific in their action, and that’s what gives it its power.
For example, they can differentiate between optical isomers and act on only one kind of
optical isomer, okay?
The enzyme hexokinase, that’s the name of the enzyme, it can act only on (plus) glucose
and not on (minus) glucose, you know, D-glucose, L-glucose, dextro (rotary) rotary, leavo rotary,
dextro rotary, leavo rotary and so on . The hexokinase can distinguish between these
two, it will act only on (plus) glucose it will not act on (minus) glucose.
It can be that specific.
And by the way, in nature in the cell, there are D-carbohydrates and L-amino acids naturally
occurring.
So L-carbohydrates and D-amino acids are not natural, usually, okay?
There are always exceptions, usually you can consider this.
So they are very specific in their action.
They act as catalysts, you all know what a catalyst does, it speeds up the rate of the
reaction several fold at that temperature pressure condition and thereby makes the reaction
possible.
Here, say, they speed of reactions several fold by decreasing the activation energy required
for the conversion of reactants to products, you all might remember the reaction coordinate
here, the energy coordinate here, the reactants are here, the products are here at a lower
energy level, there is an activation energy that needs to be crossed for this to happen.
The enzymes just bring down the activation energy as any catalyst does.
All enzymes of relevance to us are proteins and some enzymes require something called
a cofactor, such as metal ions or what are called coenzymes for their action, okay?
You have an apo-enzyme which is the proteinaceous part and to which you add a cofactor, metal
ions or coenzymes as they are called to give the whole enzyme, okay?
Just have this in mind as information, so the enzymes may not be active in the absence
of these cofactors which could just be a metal ion.
Okay?
Manganese ion.
This video, this is optional clearly this gives you the six types of enzymes.
The six types that the enzymes are classified into in a standardised fashion.
If you want you can take a look at it, it’s a lot of detail, if you’re really interested
you can go and take a look at this.
When you have an enzyme- a catalyst, it speeds up the reactions, makes reactions possible,
make cell reactions possible at the temperature pressure of the cell, which is very moderate,
right?
How do we quantify the activity of the enzyme, okay?
Its quantification is a very important aspect if you want to do something further with it,
okay?
Knowing information is fine, then we will have to find means of quantifying the information
which leads to better analysis and better manipulation application and so on so forth.
That’s why quantification becomes very important.
As engineers, some of you would know this already I just thought I will verbalise this
clearly.
The activity of pure enzymes can be quantified by something called a turnover number.
Turnover number is defined as the net substrate molecules reacted per catalyst site per time,
okay?
The way the enzyme action comes in we said was because of the three-dimensional folding.
The three-dimensional folding creates pockets that are very specific to the substrate molecule
or the reacting molecule, reactant.
The reactant comes and sits there and then it gets converted to a product by various
different means and it is the appropriate fit of the substrate molecule to the fold
in the enzyme, a part of the enzyme that makes this possible, okay?
So it is defined as the net substrate, the number of net substrate molecules reacted
per catalyst site per time, okay?
But it’s rather difficult to find out the number of molecules reacted per catalyst site
per time through regular investigations and therefore it is rarely used even in most research,
okay?
None of us use turnover number when we talk of activity of enzymes especially when we
are trying to use enzymes.
Most enzymes used in the industry are not pure, that doesn’t matter.
Their activity is expressed in terms of units of activity, right?
And this is one of the reasons why we don’t look at turnover number because you need highly
pure enzymes to even measure turnover number which itself is not very straightforward.
And on top of that you have the regular enzymes not being very pure, okay?
So we settle for something called units of activity.
Units, a Unit of activity is defined as the amount of enzyme which gives a certain amount
of catalytic activity under a prescribed set of conditions for that particular enzyme,
okay?
It is highly variable and applicable only for that particular enzyme, okay?
And people have, kind of, agreed on this.
This is how it is right now.
For example, one unit activity of glucoamylase is the amount of enzyme which produces 1 micro
mol of enzyme, 1 micro mol of glucose per minute in a 4% Lintner starch solution at
pH 4.5 and 60 degree C, okay?
(It) it can be as specific as that; amount of enzyme which produces 1 µmol of glucose
per minute in a 4% Lintner starch solution at pH 4.5 and 60 degree C. So, this is what
we use to quantify the activity of enzymes.
The speeds at which enzymes act are important to know, the kinetics is important to know
of especially for design.
Suppose we are designing an enzyme reactor to carry out an enzymatic reaction, we need
to know how long to wait for the reaction to take place if it is a batch kind of system,
or even if it’s a continuous kind of a system for design of flow rates and so on and so
forth, we need to know the kinetics.
So let us look at a simple kinetics, simple enzyme kinetics, there are very many different
kinds of kinetics, which are, which govern enzyme action; we will just look at one, for
example, okay?
This model is as follows; the enzyme reacts with the substrate to reversibly form what
is called the enzyme substrate complex, okay?
There is a reversible reaction here, forward, there is a forward reaction here, there is
a backward reaction here, there is an equilibrium at certain time and enzyme plus substrate
gives you the enzyme substrate complex.
And this irreversibly goes to give the enzyme molecule back and the product from the substrate,
okay?
This model is called the Michaelis-Menten model and it’s the simplest model in terms
of enzyme kinetics.
If we work out the rates by using this model and material balance considerations, okay,
it’s a reasonable derivation, about two pages long and if we (plot) if we look at
how the variation is with the substrate concentration, variation of the rate of the reaction with
substrate concentration, it will be something like this.
It’s a rectangular hyperbola, okay?
It is reasonably linear here, then it goes like this and asymptotically reaches what
is called a Vm, Vmax.
Mathematically, this kind of behaviour can be represented as V equals to some VmS by
Km plus S. This kind of a formulation would yield this behaviour.
So VmS by Km plus S is a good rate expression that one can choose as a first approximation
for enzyme kinetics, okay?
This is of course, this is a first approximation, this is called the Michaelis-Menton model.
Now, when the substrate concentration becomes equal to Km, let us see what happens to this
expression, right?
If it becomes equal to Km, v equals to VmS by Km plus S , so Km is S, so let’s substitute
S for Km, so you get VmS by 2S, S plus S, 2S and S and S can cancel out and you get
Vmax by 2 or Vm by 2, okay?
What does this mean graphically?
At S equals Km, the V that you find, would be Vmax by 2, okay?
So Km is called the substrate concentration which gives half maximal rate of the reaction,
right, and Km is the Michaelis-Menton constant.
So this is a nice expression to know, the first approximation to know for enzyme kinetics,
and it is useful in many different ways, I just mentioned one.
To find out, Vm and Km, as you can realise Vm and Km will be dependent on the enzyme
of the substrate and for each combination there will be a Vm and a Km, so how do you
find that out, how do you find out these model parameters here, Vm and Km.
To find that out, what we do is something like this, one of the things that we can do
is something like this because we’ll have to find that out from data.
These model parameters can be determined from the substrate versus time, the variation of
substrate concentration versus time data, you do an experiment (you) we carry out an
enzymatic reaction and then we monitor the substrate concentration with time and that’s
the data that we have, we have the substrate concentrations at various times.
From that data, how do we get the model parameters, is what we are going to look at now, at least
a basis of that.
The basis is as follows; the Michaelis-Menton is V equals to VmS by Km plus S. If we invert
this, you know, invert the left hand side and therefore we invert the right hand side,
we get 1 by V equals to Km plus S by VmS, just inversion, so if we separate out the
terms, this is a plus term here, therefore if we separate out the terms, this term would
become Km by Vm into 1byS and this term, S and S would cancel out and we get 1by Vm,
okay?
So this is of the form y equals to mx plus C or if we plot 1by V versus 1by S we expect
to get a straight line with Km by Vm as a slope and 1 by Vm as the intercept.
Such a plot is called the Lineweaver-Burke plot, plot of 1 by V versus 1 by S, you calculate
velocity from the substrate concentrations and then plot 1 by V versus 1 by S, you get
Km by Vm as a slope, and 1 by Vm as the intercept.
And I think this audience would know the slope and intercept, right?
There is the equation of a straight line y equals mx plus c, and m is the slope, c is
the intercept that’s we tried to plot it this way, so that from a straight line we
can get these parameters.
So from S versus t data calculate V using two successive datapoints S, of S and assign
it to the midpoint of the time interval there and then 1 by V versus 1 by S, straight line,
Km by Vm as a slope and 1 by Vm as the intercept.
Other types of plots can be used, there are some difficulties with this in the sense that
the data here is not very reliable and it will determine the slope and so on.
Let’s not get into that, this is an introductory course.
So this is one way of quantifying enzyme kinetics.
We talked of enzymes in the cell and how to quantify the enzyme activity and how to get
how to represent the kinetics mathematically and how to get those parameters, the model
parameters.
Now let us look at the industrial uses of enzymes, they are very widely used, f you
look at this video, it’ll tell you the various ways or some of the ways in which enzymes
are used and their usage is billions of dollars per year kind of level.
For example, if you, okay let me just point this out to you.
The industrial enzyme is video number 16, you might want to click on that and take a
look at that.
That’s this video, okay?
Please do that.
Enzymes, as we said usually proteins, are actually produced by microorganisms in bioreactors
and that is pretty much a source of enzymes nowadays, okay?
The other sources such as you know earlier there used to be other sources such as animals
and so on.
They are not very extensively used nowadays, most enzymes are produced by microorganisms
in bioreactors.
For example, the detergent industry uses proteases and amylases for better cleaning power.
Baking industry uses amylases; brewing industry uses amylases, glucanases and proteases; dairy
industry, rennin, lipases, lactases; starch industry uses amylases, glucose isomerase;
textile industry uses amylase; leather industry, trypsin, protease and cocktails of enzymes
and so on; pharmaceuticals, trypsin and so on, okay?
So, heavily used as I said billions of dollars worth per year is what what is involved, the
amount of money involved in enzyme use; enzyme manufacture, enzyme use.
So this video gives you some idea as to how enzymes are used in detergents, please take
a look at this video, it’s by novozymes but look at the the details in that video.
Also have in mind our disclaimer for all these recommendations.
When we meet up next, this is the story that we are going to ask in the larger story of
curd making, how are proteins and enzymes made in the cell, okay?
We saw that enzymes are being used, heavily used in the cell, for catalysing various different
reactions and so on, enzymes produced by microorganisms are used in everyday products and so I mean,
that’s a very large industry, the next story we’re going to see how the proteins and
the enzymes are actually made in the cell, okay?
Let’s meet up later.
See you then.