Certain cytochrome P-450s involved in the transformation of steroids catalyse not only the hydroxylation process associated with the group of enzymes, but also an acyl-carbon cleavage reaction. The hydroxylation occurs using an iron-monooxygen species while the acyl-carbon cleavage has been suggested to be promoted by an iron peroxide. In this paper we have studied the role of active site protic residues, Glu305 and Thr306, in modulating the two activities. For this purpose, the kinetic parameters for the hydroxylation reaction (pregnenolone → 17α-hydroxypregnenolone) and two different versions of acyl-carbon cleavage (17α-hydroxypregnenolone → dehydroepiandrosterone and 3β-hydroxyandrost-5-ene-17β-carbaldehyde → 3β-hydroxyandrost-5,16-diene+androst-5-ene-3β,17α-diol) were determined using the wild-type human CYP17 and its eight different single and double mutants. In addition the propensity of the proteins to undergo a subtle rearrangement converting the 450 nm active-form into an inactive counterpart absorbing at 420 nm, was monitored by measuring the 
of the P-450 → P-420 conversion. The results are interpreted to draw the following conclusions. The functional groups of Glu305 and Thr306 do not directly participate in the two proton delivery steps required for hydroxylation but may be important participants for the provision of a net work of hydrogen bonds for ‘activating’ water that then acts as a proton donor. The loss of any one of these residues is, therefore, only partially debilitating. That the mutation of Thr306 impairs the hydroxylation reaction more than it does the acyl-carbon cleavage is consistent with the detailed mechanistic scheme considered in this paper. Furthermore attention is drawn to the fact that the mutation of Glu305 and Thr306 subtly perturbed the architecture of the active site, which affects the geometry of this region of the protein and therefore its catalytic properties.
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