MEDIUM-LONG-CHAIN CHIMERIC HUMAN ACYL-COA DEHYDROGENASE - MEDIUM-CHAIN ENZYME WITH THE ACTIVE-CENTER BASE ARRANGEMENT OF LONG-CHAIN ACYL-COA DEHYDROGENASE

The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at posit ion 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA d ehydrogenase (MCADH). In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying o ut the same function is placed at position 255 on the adjacent helix G . These glutamates thus act on substrate approaching from two opposite regions at the active center. We have implemented the topology of LCA DH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu. The resulting chimeric enzyme, ''medium-/long'' chain acyl-CoA dehydr ogenase (MLCADH) has similar to 20% of the activity of MCADH and simil ar to 25% that of LCADH with its best substrates octanoyl-CoA and dode canoyl-CoA, respectively. MLCADH exhibits an enhanced rate of reoxidat ion with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA. This is the same maximum a s that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA). The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376. The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, howeve r, much narrower. The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and react ivity in acyl-CoA dehydrogenases. The mechanisms underlying these effe cts are discussed in view of the three-dimensional structure of MLCADH , which is presented in the accompanying paper [Lee et al. (1996) Bioc hemistry 35, 12412-12420].