>>Dr. Paul Sims: Welcome to this segment of the chemistry of beer. In this segment we
will discuss two enzymes, alpha-amylase and beta-amylase, that are the main ones that
are responsible for converting much of the stored carbohydrate in barley seeds into a
form that can be used by yeast. To overview what we will discuss, we will first of all
briefly touch on malting and mashing. Then we'll talk about some of the molecular properties
of starch. Some of this material will be review, but I think that's a good thing because it
helps to reinforce important concepts. Then we'll move on to talk about the properties
of alpha-amylase and beta-amylase. Our next topic will be talking about sugar transport
in yeast, and finally we’ll tie up some loose ends and summarize our discussion. This
figure shows a barley seed. In particular, notice that the starchy endosperm takes up
most of the seed. During malting the barley is allowed to germinate for a brief period
of time, and this process induces the scutellum and the aleurone layer to secrete various
hydrolytic enzymes.
During mashing, the malted barley is milled and mixed with warm water. The hydrolytic
enzymes, especially alpha and beta-amylase, continue to break down the starchy endosperm.
To remind ourselves of the molecular properties of starch, we first of all have to start with
the building block, which is glucose. As you may recall from the carbohydrate discussion,
glucose can exist in a linear form or in two different cyclic forms, which we call pyranose
forms, an alpha-D-pyranose form and a beta-D-pyranose form. The cyclic or pyranose forms are the
result of an intramolecular reaction that forms a hemiacetal. Because the nucleophilic
attack of the carbon five, or C-5 hydroxyl, on the aldehyde carbon or C1, which is also
called the anomeric carbon--because this attack can occur from above or below the plane of
this group, there are two possible cyclic forms: the alpha and beta forms. If you look
at our diagram, you'll see that the beta form has a hydroxyl group in the equatorial position,
and the alpha form has the hydroxyl group is the axial position. A mnemonic device to
help you remember and distinguish between alpha and beta is “batter up for beta”
if that helps. It is important to keep in mind that the conversion between the linear
form and the cyclic forms is a dynamic process. The equilibrium greatly favors the cyclic
forms, and in particular the beta form. The reason why the beta form predominates is because
the C1 hydroxyl experiences less steric hindrance in the equatorial position. However, it's
possible to introduce a reagent called Fehling’s reagent or Benedict's reagent that will oxidize
the aldehyde carbon or carbon(I), the anemeric carbon. In the process one of the components
of the Fehling’s reagent or Benedict's reagent copper(II) ions gets reduced to copper(I).
So as the glucose aldehyde group is oxidized to a carboxylate, the copper(II) is reduced
to copper(I). Copper(I) forms part of an insoluble precipitate that’s a red color, and this
forms the basis of a qualitative test to detect the presence of a reducing sugar.
Recall that glucose molecules can join together in a condensation or dehydration reaction.
We refer to the glucose molecules that have been incorporated into a polymer as glucose
residues because water has been lost when the two glucose molecules were joined. Below
is an example of two glucose molecules condensing to form what we call an alpha-1,4 linkage.
The resulting disaccharide is maltose, which you will hear more about during this segment.
We call the linkage an alpha-1,4 linkage because the hydroxyl that is attached to one of the
glucose molecules is in the alpha position. Remember “batter up beta.” Therefore,
alpha is below the plane of the ring so an alpha-1 linkage connects the number 1 carbon
of one glucose with the number 4 carbon of the other glucose, and it’s alpha because
the group was in the alpha position prior to the loss of water.
Previously you were introduced to amylose, so here's a reminder of the structure of amylose.
It is essentially a linear polymer of glucose residues connected by alpha-1,4 linkages.
The number of glucose residues in an amylose polymer is often in the thousands. Now we
talked about the fact that glucose is a reducing sugar, and when we connect several glucose
residues together to make amylose, we have a reducing end, where the number 1 carbon
would still be capable of opening to the aldehyde and therefore still be capable of being oxidized
to the carboxylate. And then we have a non-reducing end, where we end with the number 4 carbon,
and the anemeric carbon is tied up in a glycosidic bond, and it is not capable of opening up
to be oxidized to the aldehyde, so we refer to that end of amylose as the non-reducing
end.
Amylopectin, as you may recall, is also a polymer of glucose residues, and these glucose
residues are connected predominantly by alpha-1,4 linkages, but they also have alpha-1,6 branch
points. The branch points occur every 24 to 30 residues, and the molecular weight of amylopectin
can be up to 100 million. If you look at our diagram, you will see that many of the linkages
are alpha-1,4, similar to what we saw with amylose, but I'm showing one alpha-1,6 branch
point. We connect the number 1 carbon, which had the hydroxyl group in the alpha position--that's
why we call it the alpha-1--to 6 because it's connected to the 6 carbon in the chain below
and that forms our alpha-1,6 branch point.
So now that we’ve discussed the properties of amylose and amylopectin, the major components
of the starchy endosperm in barley seeds, we are now ready to talk about the enzymes
that catalyze hydrolysis reactions involving these two polymers. The two main enzymes,
as we said, are alpha-amylase and beta-amylase. They’re going to break down the starchy
endosperm in barley seeds and make this stored carbohydrate into a form that the yeast can
then use. Both enzymes have a pH optimum of about 5 and a relatively high temperature
optimum, 70° and 60° respectively for alpha-amylase and beta-amylase. This explains why the mashing
process uses warm water because they want to have the temperature to matching the optimum
temperature of these two enzymes. Alpha-amylase is a monomeric enzyme, and what we mean by
that it is made up of one subunit. The subunit has a molecular weight of 45,000. It requires
calcium ions for activity, and it randomly catalyzes the hydrolysis of alpha-1,4 glycosidic
linkages, but not near branch points or at non-reducing ends of amylose or amylopectin.
Beta-amylase also is a monomeric enzyme. It has a slightly higher molecular weight of
60,000. There are two active site glutamates that are thought to participate as general
acid-based catalytic residues. Beta-amylase catalyzes the hydrolysis of alpha-1,4 glycosidic
linkages near the non-reducing ends of amylose and amylopectin, such that maltose residues
are released.
Now I realize this is a lot of verbiage, and it'll help to have a picture processes in
mind. If we look at this diagram, this schematically shows where beta-amylase catalyzes hydrolytic
cleavages of both amylose and amylopectin, and it's trying to convey how alpha-amylase
catalyzes an internal cleavage. And it happens at random, just does not happen too near to
branch points or too near to non-reducing ends. But through the combined reaction of
these two enzymes, the relatively long polymers of amylose and amylopectin are broken down
into simpler sugars that the yeast can use. Shown in these two figures are alpha-amylase
and beta-amylase. In particular, note the calcium ions, which are represented as green
spheres that alpha-amylase requires, and also note the catalytic glutamates, which are shown
in stick form, that beta-amylase uses as part of its catalytic residues. If you want, you
can view and explore these and other structures using the program UCSF chimera. You can find
this program at the indicated URL. Once you’ve downloaded this program, you can visit the
PDB, or protein databank, and obtain the codes for these two structures or other proteins
that you’re interested in. And you can view these structures and manipulate these structures
on your home computer, and I strongly encourage you to do so.
So let’s compare the breakdown of amylose and amylopectin with glycogen breakdown in
humans. Our storage form of glucose is called glycogen. It is similar to amylopectin, but
the alpha-1,6 branch points occur more frequently, about every 8 to 12 residues. The enzyme glycogen
phosphorylase catalyzes the phosphorolysis of alpha-1,4 linkages from the non-reducing
ends of glycogen as indicated in the figure below. We start with glycogen, n residues
long, and instead of water, we use HPO42-. That’s inorganic phosphate. So instead of
a hydrolysis reaction, we have a phosphorolysis reaction, and what we form is glucose-1-phosphate.
And we still have our glycogen, but it's minus a residue. Now this reaction saves an ATP
because the glucose-1-phosphate can be converted to glucose-6-phosphate, and this will be clearer
to you when we have our discussion on glycolysis.
But here's the question: why would this reaction not be helpful to yeast that are relying on
sugar from the starchy endosperm of the barley seed? To answer this question, we have to
think about transporters in yeast, sugar transporters in yeast. Shown on the left is a glucose transporter.
This the glucose transporter is an integral membrane protein. It facilitates the diffusion
of glucose from outside the cell into the cell. However, if the glucose has been phosphorylated,
as in glucose-1-phosphate, it is no longer able to pass through the glucose transporter.
So if the barley enzymes broke down the amylose and amylopectin in such a way that we formed
glucose-1-phosphate, then that sugar, that phosphorylated sugar would not be available
to the yeast, but free glucose can diffuse into yeast. And importantly, shown on the
right, maltose can diffuse into yeast, and this transporter is a little bit different.
Notice that along with the maltose coming into the cell, we have a hydrogen ion coming
into the cell. This process is called symport. This is a symporter. Once the maltose is inside
the yeast, an enzyme called maltase can catalyze the hydrolysis into two glucose molecules.
Once the glucose has entered the cell, either as free glucose or as maltose, which is then
hydrolyzed by maltase into two glucose molecules, the glucose can then enter glycolysis and
ultimately produce the ethanol that we find in beer.
So now we’re ready to summarize our discussion. There are other hydrolytic enzymes, including
a number of proteases that are important in the brewing process, but we decided to focus
on the major ones, alpha-amylase and beta-amylase. Alpha-amylase randomly catalyzes the cleavage
of alpha-1,4 glycosidic linkages, but not near branch points or at non-reducing ends
of amylose or amylopectin. Beta-amylase catalyzes the hydrolysis of alpha-1,4 glycosidic linkages.
These happen at the non-reducing ends, and they happen in such a way that maltose is
released from the non-reducing ends. Mashing is done at a temperature and pH that are close
to the optimum levels for these two enzymes, and it is through the combined action of these
two enzymes that much of the carbohydrate in the starchy endosperm is made available
to yeast. So that concludes our discussion of alpha-amylase and beta-amylase. You will
see in future segments how all of these concepts come together, and you’ll appreciate the
unified applied biochemistry that is brewing beer.