Evidence for the existence of the enzyme substrate complex-
1) At constant concentration of enzyme the rate of reaction increases with increased concentration of substrate (up to Vmax). Uncatalyzed reactions do not show a maximal velocity and hint at the formation of an enzyme substrate complex. Vmax occurs when all the enyme molecules are saturated with substrate.
2) X-ray crystallography has shown high resolution structures of enzymes catalysing reactions with substrates trapped in the active site in various conformations. These trapped substrates in stressed conformations represent the transitions state at which the activation energy for conversion of substrate to product is lowest.
3) Kinetic studies show that enzymes have a high degree of specificity and only catalyse a very limited number of reactions each (usually 1). This high specificity is determined by a well formed active site where substrate binds. Substrate analogues have been shown to bind to active sites (by crystallography and spectroscopy) confirming the idea of a docking site for substrate that complements its 3D shape (inhibition studies).
4) Linus Pauling suggested “The attraction of the enzyme molecule for the activated complex would thus lead to a decrease in its energy and hence to a decrease in the energy of activation of the reaction and to an increase in rate of reaction” The idea of the enzyme active site complementing the transition state of the active site is supported by crystallographic studies and kinetic studies, as well as NMR studies.
DNA MTases (methyltransferases) catalyse the transfer of methyl groups to DNA from AdoMet (S-adenosyl-L-methionine) producing AdoHcy (S-adenosyl-L-homocysteine) and methylated DNA. The C5 and N4 positions of cytosine and N6 position of adenine are the target sites for methylation. All three methylation patterns are found in prokaryotes, whereas cytosine at the C5 position is the only methylation reaction that is known to occur in eukaryotes. In general, MTases are two-domain proteins comprising one large and one small domain with the DNA-binding cleft located at the domain interface. The striking feature of all the structurally characterized DNA MTases is that they share a common core structure referred to as an ‘AdoMet-dependent MTase fold’. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate (covalent attachment of the enzyme to the DNA). oligodeoxynucleotides containing 5-fluorodeoxycytidine have been used as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA.
• Glutamate forms 2 hydrogen bonds with cytosine at N3 and the amide H linked to C4
• Hydrogen is transferred from Glu to N3 allowing cysteine to perform nucleophilic attack on C6
• S-adenosyl-L-methionine donates a methyl onto C5
• Glu is re-protonated and cysteine is released, creating 5-methyldeoxycytidineand S-adenosyl-L-homocysteine.
In the case of C5-cytosine MTases (Figure 2A), it was shown that motif IV which possesses an invariant Pro-Cys dipeptide was involved in methyl group transfer [10,91,92]. Mutagenesis of this dipeptide, leading to loss of function in M.EcoRII, M.HhaI and M.HaeIII, was one of the several criteria proposed for the importance of the Pro-Cys dipeptide sequence in motif IV
The cysteine has already been characterised at the molecular level within the mechanism of C5 methly transfer, but further site direct mutagenesis studies could improve our understanding ( and possibly mislead us). The conserved proline that precedes the cysteine is an interesting target for mutagenesis, proline create kinks in alpha helices and there presence usually infers an important structural component. Switching the proline for an amino acid that doesn’t function as a helix breaker such as alanine, could give us important information on the angle of the covalent bond with cytosine using circular dichroism spectroscopy. The cysteine itself could be the target of site directed mutagenesis, but as no other natural amino acid offer its special chemical properties (sulphur) it is unlikely a valid confer will restore activity. insertion of a Ser residue between the Pro-Cys motif renders the enzyme inactive this proves that the angle created by the proline is important but also it’s main chain carbon or the cysteines position is critical. Site directed mutagenesis produces radical results but the actual relative usefulness of them can be rather minimal. What does deleting/altering a key residue and recording minimal/no activity really tell you?