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[求助]
【求助】+有关酶与底物的问题
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会做一部分但是不确定呀。第1问不明白，3不确定用什么方法。第一问主要讲：为什么这个酶在它的基底附近的区域显示出氨基酸的高保守度（和这个酶family的）？下面是原题，其实很好懂的，就是需要些耐心，麻烦大家了！给些重要提示就行呢，完全没有思路呀。。。就告诉我第一问也行呀，拜托了，好郁闷。。。 Examine the crystal structure in figure 1 below of the enzyme, Alpha, an essential enzyme of DNA metabolism from a human parasite. This enzyme is found in all eukaryotic genomes that have been sequenced. The residues in the structure are shown with their electron density to give an indication of the true 3-dimensional shape of the protein. The residues are coloured according to their degree of conservation within the eukaryotic Alpha-protein family. Absolutely conserved amino acids shared by all Alpha-like enzymes are coloured in the darkest shade of grey. Amino acids, with lesser degrees of conservation, are indicated by lighter grey shading, whereas those amino acids with no homology are coloured white. The position of the bound substrate for enzyme Alpha is indicated. (a) Explain, with reference to the above figure why the area around the substrate shows the greatest degree of amino acid conservation within the Alpha enzyme family ================= Further structural analysis of enzyme Alpha reveals a binding site for the drug that is known to selectively target the parasite enzyme, see figure 2 below. Intriguingly neither enzymes Beta (human) or Gamma (mouse) are affected by the inhibitor molecule that is found to bind to enzyme Alpha (parasite), despite considerable sequence homology in this region of the three proteins. It appears that the inhibitor does not bind to these enzymes. The company developing the inhibitor for use against the parasite are unable to crystallise the human or mouse enzymes to understand the basis for the selective inhibition of the parasite enzyme. You are asked by the company developing the inhibitor to deduce the likely basis for the selective binding to the parasite enzyme. You decide to align the eleven amino acid region where the enzyme Alpha binds the inhibitor molecule with the same regions of enzymes Beta and Gamma, shown in figure 3, below. 1 5 10 Enzyme Alpha (parasite) SGDLVRGKEGR Enzyme Beta (human) TYNVIRGDNYR Enzyme Gamma (mouse) TGDIVRGENFR Figure 3. Amino acid alignment of the parasite drug binding site Alignment of homologous region to parasite drug binding site in the human and mouse proteins, numbering is for the parasite sequence arbitrarily beginning at residue 1. (b) Suggest, with reasons, which amino acids may be involved in binding the inhibitor and thus explain why enzymes Beta and Gamma do not bind the inhibitor molecule. (c) Write a brief outline of how you would assay the role of the residues in Alpha or Beta to confirm your hypothesis about which residues are responsible for inhibitor binding. You may assume that a simple, quantitative in vitro assay is available to monitor enzyme activity, that all three enzymes are cloned into expression plasmids and that soluble forms of the proteins can be prepared easily from the bacterium E.coli. (d) The human homologue of the parasite protein functions as a monomer of 48 kDa. It also exists as a tetramer whose role is not known but appears to be functionally inactive. It does not seem to be post-translationally modified when isolated from human tissue. On incubation of the tetramer with magnesium it dissociates into the metal-dependent active form. Outline how would prepare a sample of the protein for crystal trials. You may take advantage of whatever technology you are familiar with.   |
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