Contributions of Q67 and Y69 Residues to Ligand Binding and Catalysis in R67 Dihydrofolate Reductase

Dihydrofolate reductase (DHFR) serves an important role in metabolism by reducing dihydrofolate (DHF) to the product tetrahydrofolate via hydride transfer from NADPH. R67 DHFR, a plasmid encoded form of the enzyme which provides resistance to trimethoprim, functions as a homotetramer with D2 symmetry. Both ligands, DHF and NADPH, interact within a 25 Ǻ active site pore. Mutagenesis of one active site residue results in four-symmetry related mutations causing large effects on binding and catalysis. A construct containing four copies of the DNA for R67 DHFR ligated in-frame and flanked by unique restriction sites was engineered and asymmetric mutants were built using this construct. Q67H asymmetric mutants were built with the goal of preserving tight binding without inhibition, as Q67H R67 DHFR binds both DHF and NADPH with greater affinity than the wild-type enzyme, but also yields severe DHF and NADPH inhibition [Park, H., Bradrick, T. D., and Howell, E. E. (1997) Protein Eng. 10, 1415-1424]. Although many of the Q67H asymmetric mutants bind NADPH with greater affinity than the control, inhibition is often observed. From these studies, a role for Q67 in selecting for the productive ternary complex over inhibitory complexes was proposed. Asymmetric Y69F mutants were also generated, as the kcat for Y69F R67 DHFR is increased 2 fold compared to the wild-type enzyme, while the Km values are increased [Strader, M. B., Smiley, R. D., Stinnett, L. G., VerBerkmoes, N. C., and Howell, E. E. (2001) Biochemistry 40, 11344-11352]. These asymmetric mutants were constructed with the goal of increasing kcat while maintaining high affinity. Although this goal was not accomplished, these asymmetric mutants provided insight into ligand binding and catalysis in R67 DHFR as they support a model where two Y69 residues interact with NADPH, while mutations along the dimer-dimer interface increasing kcat. Thus, generating asymmetric mutants of R67 DHFR has provided a means by which to understand ligand binding and catalysis in a homotetrameric enzyme where only a single active site pore is available

Mutagenesis of rat acyl-CoA synthetase 4 indicates amino acids that contribute to fatty acid binding

Although each of the five mammalian long-chain acyl-CoA synthetases (ACSL) can bind saturated and unsaturated fatty acids ranging from 12 to 22 carbons, ACSL4 prefers longer chain polyunsaturated fatty acids. In order to gain a better understanding of ACSL4 fatty acid binding, we based a mutagenesis approach on sequence alignments related to ttLC-FACS crystallized from Thermus thermophilus HB8. Four residues selected for mutagenesis corresponded to residues in ttLC-FACS that comprise the fatty acid binding pocket; the fifth residue aligned with a region thought to be involved in fatty acid selectivity of the Escherichia coli acyl-CoA synthetase, FadD. Changing an amino acid at the entry of the putative fatty acid binding pocket, G401L, resulted in an inactive enzyme. Mutating a residue near the pocket entry, L399M, did not significantly alter enzyme activity, but mutating a residue at the hydrophobic terminus of the pocket, S291Y, altered ACSL4’s preference for 20:5 and 22:6 and increased its apparent Km for ATP. Mutating a site in a region previously identified as important for fatty acid binding also altered activation of 20:4 and 20:5. These studies suggested that the preference of ACSL4 for long-chain polyunsaturated fatty acids can be modified by altering specific amino acid residues