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active site
Reactive groups in enzyme active sites are optimally positioned to interact with the substrate
active site Figure 2-22 The electrostatic potential around the enzyme Cu,Zn-superoxide dismutase Red contour lines indicate net negative electrostatic potential; blue lines net positive potential. The enzyme is shown as a homodimer (green ribbons) and two active sites (one in each subunit) can be seen at the top left and bottom right of the figure where a significant concentration of positive electrostatic potential is indicated by the blue contour lines curving away from the protein surface. The negative potential elsewhere on the protein will repel the negatively charged superoxide substrate (O2) and prevent non-productive binding, while the positive potential in the active site will attract it. Graphic kindly provided by Barry Honig and Emil Alexov.
In any enzyme-catalyzed reaction, the first step is the formation of an enzymesubstrate complex in which the substrate or substrates bind to the active site, usually noncovalently. Specificity of binding comes from the close fit of the substrate within the active-site pocket, which is due primarily to van der Waals interactions between the substrate and nonpolar groups on the enzyme, combined with complementary arrangements of polar and charged groups around the bound molecule. This fit is often so specific that even a small change in the chemical composition of the substrate will abolish binding. Enzymesubstrate dissociation constants range from about 103 M to 109 M; the lower the value the more tightly the substrate is bound. It is important that an enzyme does not hold onto its substrates or products too tightly because that would reduce its efficiency as a catalyst: the product must dissociate to allow the enzyme to bind to another substrate molecule for a new catalytic cycle. Formation of a specific complex between the catalyst and its substrates does more than just account for the specificity of most enzymatic transformations: it also increases the probability of productive collisions between two reacting molecules. All chemical reactions face the same problem: the reacting molecules must collide in the correct orientation so that the requisite atomic orbitals can overlap to allow the appropriate bonds to be formed and broken. If we consider, in general terms, that a molecule can have a reactive side where the chemical changes take place and an unreactive side where they do not (at least immediately; the
(a)
(b) (c)
+ +
+ ++
+ +
+ + +
Figure 2-23 Schematic diagram showing some of the ways in which electrostatic interactions can influence the binding of a ligand to a protein (a) Electrostatic forces and torques can steer the ligand (green) into its binding site on the protein (shown in yellow). (b) Some binding sites are normally shielded from the solvent and can be kept closed by salt links between groups on the protein surface. If the correct substrate disrupts these salt links it can gain access to the binding site. This is known as gated binding. Alternatively, the dynamics of the protein may open and close such a site transiently (as indicated by the yellow arrows). (c) Electrostatic interactions, particularly salt links and hydrogen bonds, between ligand and protein can contribute to the affinity and specificity of binding and to the orientation of the ligand in the binding site and the structure of the complex formed. All three of these ways of exploiting electrostatic interactions can be used by a single enzyme. Adapted from Wade, R.C. et al.: Proc. Natl Acad. Sci. USA 1998, 95:59425949.
Definitions gated binding:binding that is controlled by the opening and closing of a physical obstacle to substrate or inhibitor access in the protein. reaction sub-site: that part of the active site where chemistry occurs. specificity sub-site: that part of the active site where recognition of the ligand takes place.
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aspartate
Asp 222
Figure 2-24 Schematic diagram of the active site of E. coli aspartate aminotransferase The enzyme uses a pyridoxal phosphate (PLP) cofactor (purple) and lysine (yellow outline) to carry out chemistry. The substrate amino acid (green) reacts with the cofactor to form an adduct (as shown in this model) which then rearranges to give product. Substrate specificity for the negatively charged aspartic acid substrate is determined by the positively charged guanidino groups of arginine 386 and arginine 292, which have no catalytic role. Mutation of arginine 292 to aspartic acid produces an enzyme that prefers arginine to aspartate as a substrate. Adapted from Cronin, C.N. and Kirsch, J.F.: Biochemistry 1988, 27:45724579; Almo, S.C. et al.: Prot. Eng. 1994, 7:405412.
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