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Extracellular Polysaccharides & Caries

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Part 1 Some basic sugar chemistry

Part 2 The structure of the polymers

Part 3 The reactions catalysed by FTFs and GTFs (this page)

Part 4 The enzymes themselves

Part 5 The role of EPS in dental plaque and caries

Part 3 - The enzyme reactions

The reactions catalysed

At its simplest the reaction involves the hydrolysis of sucrose and the concomitant addition of a glucose or fructose residue to a growing polymer. The release of the remaining sugar moiety of sucrose is a by-product of the reaction. The reaction below shows the addition of glucose to a growing mutan polymer but the reaction for the other enzymes could be written similarly.

Source of the energy

This is one of the more obvious questions. Normally, biosynthetic pathways utilise energy-rich intermediates. Sometimes this simply means that a reaction substrate is phosphorylated by the expenditure of ATP and sometimes the substrate is covalently bonded to a phosphorylated base such as uridine diphosphate. In either case some energy is added to the system in the form of a chemical bond involving phosphate. This is then used as the source of energy to drive the synthetic reaction.

Cells export material all the time and not all of it is waste product. Enzymes such as proteases and polysaccharides such as bacterial capsular substances are good examples. These all represent energy because energy was required to synthesise them. However, incorporating mechanisms into cell membranes and walls which secrete high-energy anabolic intermediates such as ATP into the environment would be analogous to someone scattering money around a street and expecting it to be there when they needed it.

The answer is that the energy required to synthesise EPS comes from sucrose itself. The energy required to form a glycosidic bond of the type used in EPS is about 13.5 kJ/mol (3.2kcal/mol). The energy in the glycosidic bond between glucose and fructose in sucrose is about 29 kj/mol (7 kcal/mol) which, given that some energy is always lost during reactions, is sufficient for the synthesis.

sucrose

Glycogen synthesis is comparable to EPS synthesis but takes place intracellularly. In this reaction the energy required to form the glycosidic bond which adds a glucose residue to the growing glycogen polymer is contained within the high-energy intermediate UDP-glucose.

What is so special about sucrose?

Sucrose differs from many other disaccharides in the type of glycosidic bond which links the two hexoses. In disaccharides such as maltose and lactose the glycosidic bonds link carbons C1 and C4. In all aldose sugars, C1 is the carbon with the aldehyde group attached and is known as the anomeric carbon atom because this structure allows for two different configurations known as anomers . A similar situation is found in ketose sugars except the anomeric carbon atom is C2.

The free energy associated with the glycosidic bond in maltose is 16.7 kj/mol (7 kcal/mol) whereas the free energy associated with the glycosidic bond of sucrose is 29 kj/mol (7 kcal/mol)l. The reason for this is that the glycosidic bond of sucrose links both anomeric carbon atoms whereas the bond in maltose links the anomeric carbon of one glucose with C4 of another. In lactose the glycosidic bond links the anomeric carbon, C1, of galactose with C4 of glucose.

The free energy associated with the glycosidic bond of sucrose is, therefore, equivalent to that of phosphorylated precursors used in intracellular synthetic pathways and is sufficient to provide 13.5 kJ/mol (3.2kcal/mol) required to sequentially add glucose or fructose units to the growing EPS chain. Insufficient energy is released by the hydrolysis of, say, maltose because significant energy is lost during the process.

Priming the reaction

Early researchers studying these enzymes naturally looked at what we then knew about similar polysaccharide-synthesising enzymes such as that responsible for the formation of glycogen. In this case the reaction requires a priming molecule in the shape of some pre-existing glycogen. When they looked at the effect of various pre-formed glucans on different GTFs they got some strange results.

They found that GTFs are active in the absence of exogenous glucan which suggests a primer or reaction initiator is not required. However they also found that exogenous glucan can activate or speed up the action of some GTFs.

We now have evidence that exogenous glucan speeds up these GTFs by binding to a site remote from the catalytic site which causes a conformational change in the enzyme and speeds it up. GTFs are, therefore, very different enzymes to glycogen synthase.

The reaction mechanism

It is a popular misconception that enzymes do one job and do it in just one direction. In nearly all cases this is not correct. GTFs and FTFs are no exception and a glucan or fructan, of whatever variety, is not the only possible product of a reaction with sucrose.

Apart from the addition of a glucose or fructose residue to the growing polymer there are 3 other possible reactions.

Possibility 1 Nothing changes.

Sucrose reforms and dissociates from the enzyme-substrate complex. This, actually is a very common outcome for all enzyme reactions.

Possibility 2 Sucrose is hydrolysed

The enzyme-bound glucose (GTF) or fructose(FTF) is added to water. The outcome of the reaction is then merely the hydrolysis of sucrose into glucose and fructose. Actually this is an example of an acceptor reaction (see next) with water acting as the acceptor molecule.

Possibility 3 The acceptor reaction

The enzyme-bound glucose or fructose is added to an acceptor molecule.

In the case of GTFs, the acceptor molecule can be glucose, a small saccharide such as maltose, isomaltose or, indeed, sucrose itself. In the presence of such an acceptor molecule and sucrose (as substrate) GTFs switch from glucan synthesis and start making oligosaccharides (short chain polymers up to 20 residues long) but with the same bond specificity of the GTF. Pre-formed glucans can also act as acceptors as can fructose if it is present in high enough concentration. In this case an unusual disaccharide called leucrose is formed.

FTFs are similar. They can use sucrose or pre-formed fructans as an acceptor.

This is important because ..

It is far too simplistic to think that within dental plaque some sucrose is converted into glucan, mutan, levan or inulin-like polymer and that these are more or less important in caries.

Dental plaque is very complicated. It is also dynamic which means that it is constantly changing. A large number of different bacterial species are present some of which make different glucose and fructose polymers. Also present are lots of different saccharides such as maltose and dextrins which we know through studies of isolated enzymes can take part in the acceptor reaction (see opposite). In other words, they are complicating the simple view.

Knowledge of what is actually happening in the reaction with sucrose and especially the acceptor reaction phenomenon helps our understanding of the factors which affect the formation and fate of EPS so we can make sense this dynamic situation within dental plaque.

This is leucrose

This interesting sugar was discovered by researchers studying dextransucrase. It is the product of fructose acting as an acceptor molecule for GTF. Because the bond is between carbon 1 of the glucose residue and carbon 5 of the fructose residue the fructose cannot exist in its usual 5 membered furan form but has to adopt the 6 membered pyran configuration.

Polymer synthesis

Processive or not processive?

Next, it is important to know if the polymer being synthesised stays attached to the enzyme for the duration of the synthetic reaction (a processive reaction) or whether it breaks free after the addition of each sugar residue (a non-processive reaction). Why this is important is described opposite.

The generally accepted view is that the reaction is of a type somewhere between processive and non-processive. That is to say the product stays bound for a short while during which one or several residues are added to the growing polymer. The polymer then breaks free from the enzyme which then binds to a different polymer, the same site on the same polymer or a different site on the same polymer. This has been called a semi-processive reaction.

This is important because ..

Early researchers assumed that each enzyme only made one type of bond. This made it difficult to explain how branched polymers were formed unless the polymer broke free and a different enzyme added branch points. Then it was discovered that, in fact, some of these GTFs and FTFs could actually synthesise two types of bond. Quite how this happens or is controlled is not really understood yet.

While this explains how the structures described in Part 2 could be synthesised it does not explain the more complex, cross-linked polymers we know exist in the matrix of dental plaque. The semi-processive mechanism described opposite can help with this.

Reducing or non-reducing end?

The next big issue is whether the enzyme adds residues to the reducing end or the non-reducing end of the forming polymer. In the case of glucans, we need to know if the chain is extended by adding a glucose to Carbon 1 of the terminal glucose or Carbon 3 or 6 of some other glucose?

Similarly for fructans but with these polymers reducing terminus is Carbon 2

This has been looked at extensively and the weight of evidence favours additions at the non-reducing end.

The reducing end is the end where the anomeric carbon atom (C1 for glucose and C2 for fructose) is free and has not been used to form a glycosidic bond. Polymer elongations to C1 are described as being at the reducing end. Elongations made by additions to any other carbon atom are to the non-reducing end.

SUMMARY
1	Both glucose and fructose polymers are formed by additions to the non-reducing ends of the elongating polymers in a semi-processive manner.
2	Different GTFs and FTFs are made which can catalyse the formation of one or more different glycosidic bonds.
3	The catalytic reaction is semi-processive and because of this the enzyme can use pre-existing glucans or fructans as the acceptor molecule.
4	In the case of glucan synthesis this also means that pre-formed glucans can be modified by a GTF which was not initially resposnible for its formation. This ability to modify existing polymers can lead to highly complex, cross-linking.
5	This doesn't seem to happen with fructans even though fructose is randomly added to existing polymers. The most likely explanation is that FTFs are more specific in their acceptor recognition.

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