Redox properties of human medium-chain acyl-CoA dehydrogenase, modulation by charged active-site amino acid residues.
The modulation of the electron-transfer properties of human medium-chain acyl-CoA dehydrogenase (hwtMCADH) has been studied using wild-type and site-directed mutants by determining their midpoint potentials at various pH values and estimating the involved pKs. The mutants used were E376D, in which the negative charge is retained; E376Q, in which one negative charge (pKa approximately 6. 0) is removed from the active center; E99G, in which a different negative charge (pKa approximately 7.3) also is affected; and E376H (pKa approximately 9.3) in which a positive charge is present. Em for hwtMCADH at pH 7.6 is -0.114 V. Results for the site-directed mutants indicate that loss of a negative charge in the active site causes a +0.033 V potential shift. This is consistent with the assumption that electrostatic interactions (as in the case of flavodoxins) and specific charges are important in the modulation of the electron-transfer properties of this class of dehydrogenases. Specifically, these charge interactions appear to correlate with the positive Em shift observed upon binding of substrate/product couple to MCADH [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715], which coincides with a pK increase of Glu376-COOH from approximately 6 to 8-9 [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. From the pH dependence of the midpoint potentials of hwtMCADH two mechanistically important ionizations are estimated. The pKa value of approximately 6.0 is assigned to the catalytic base, Glu376-COOH, in the oxidized enzyme based on comparison with the pH behavior of the E376H mutant, it thus coincides with the pK value recently estimated [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The pKa of approximately 7.1 is assigned to Glu376-COOH in reduced hwtMCADH. Comparable values for these pKas for Glu376-COOH in pig kidney MCADH are pKox = 6.5 and pKred = 7.9. The Em measured for K304E-MCADH (a major mutant resulting in a deficiency syndrome) is essentially identical to that of hwtMCADH, indicating that the disordered enzyme has an intact active site.
Mancini-Samuelson
G J
GJ
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Kieweg
V
V
Sabaj
K M
KM
Ghisla
S
S
Stankovich
M T
MT
D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004735:Energy Transfer; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry
D011994:Recombinant Proteins; D018698:Glutamic Acid; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D008239:Lysine; D005998:Glycine