Abstract
Site-directed mutants of sperm whale myoglobin were prepared to probe the functional role of the highly conserved distal pocket valine residue, Val68(E11). This amino acid was replaced with Ala, Ile, and Phe to examine the effects of the side chain volume at position 68 on ligand binding. Three double mutants were also constructed in which the distal His64(E7) was replaced with Gly and Val68 was replaced with Ala, Ile, and Phe to determine the effects of size at position 68 in the absence of the distal histidine. Association and dissociation rate constants for O2, CO, and alkyl isocyanide binding were measured by stopped-flow rapid mixing, conventional flash, and laser photolysis techniques at pH 7, 20 degrees C. The association rate constants for the binding of all eight ligands to the single mutants decreased in the order Ala68 greater than Val68 (native) greater than Ile68 myoglobin, indicating that the 68(E11) residue is part of the overall kinetic barrier. A similar pattern was observed for the association constants of the double mutants: Gly64/Ala68 greater than Gly64/Val68 greater than Gly64/Ile68. Thus, increasing size of the E11 side chain inhibits the rate of ligand binding even in the absence of histidine at position 64. Substitution of Ala for Val68 had little effect on O2 affinity but did increase the affinities for CO and isocyanide binding. The affinities for all of the ligands were decreased for the Ile68 mutant. The ligand binding affinities for the Gly64/Ala68, Gly64/Val68, and Gly64/Ile68 myoglobins displayed an analogous trend to that of the single mutants, indicating that the equilibrium interactions between the position 64 and 68 side chains and the bound ligand are roughly additive. Both the association rate constants and dissociation rate constants for O2 and isocyanide binding were decreased for the Phe68 mutant myoglobin. These kinetic parameters result in little change in O2 affinity and an increase in isocyanide affinity, relative to the native protein. Thus, the large benzyl side chain of phenylalanine at position 68 inhibits the rate of ligand movement up to and away from the iron atom but not the final bound state.
Highlights
Association and dissociation rate constants for 02, CO, and alkyl isocyanide binding were measured by stopped-flow rapid mixing, conventional flash, and laser photolysis techniques at pH 7, 20 “C. The association rate constants for the binding of all eight ligands to the single mutants decreased in the order Alaee > Valss > IlesS
Association Rate Constants for VuP Mutants-The observed time courses for ligand binding to the Ala@, Ile6’, and
The observed association rate constants (k’) for the Valm mutants are given in Table I, and displayed in Fig. 2A as a function of the size of the amino acid at position 68 (Ell)
Summary
This amino acid was replaced with Ala, Ile, and Phe to examine the effects of the side chain volume at position. Glye4/Ilees myoglobins displayed an analogous trend to that of the single mutants, indicating that the equilibrium interactions between the position 64 and 68 side chains and the bound ligand are roughly additive Both the association rate constants and dissociation rate constants for O2 and isocyanide binding were decreased for the Phess mutant myoglobin. These kinetic parameters result in little change in O2 affinity and an increase in &cyanide affinity, relative to the native in accordance with 18 USC.
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