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AbstractHumanaromatase(AROM) catalyzes theconversion of androgens to estrogensand is a major breast cancer drug target. Structural investigation has provided insights intothe active siteandaromatization mechanism.Utilization of the structural data has permitted rational design of a series of novel steroidal inhibitors. Investigation ofthe roles of key amino acids is facilitated by a recombinant AROM identical in crystal structureto the placental AROM.We use mutagenesis, chromatography, ultracentrifugation, spectrophotometry, enzyme kinetics, and X-ray crystallography to probe the roles of critical residues and the molecular basis of oligomerization. Furthermore, weevaluate the potencies of novel inhibitors and determine the structural basis of inhibition andselectivity. A critical active site residue D309 withan elevated pKa remainsprotonated at neutral pHand facilitatessubstrate binding and catalysis.The “gatekeeper” R192, linked to D309 via a watermolecule, is postulated to have a role in proton relay and substrate selectivity. D309N and R192Q mutants are virtually inactive supporting thehypothesis that both play keyrolesin aromatization.AROM oligomerization is driven bytheD-E loop of one moleculeand heme-proximal region of another via hydrogen bonding, electrostatic interactions between E181 and K440, and shape complementarity.Del7, generated by deletionof 7 residues in the D-E loop, experiences 65% reductionin activitydue to the loss of oligomer formation. Mutants Del4, E181A, and E181K exhibit normal enzymatic activity,and maintain some oligomeric interactions. The heme-proximal interface is also the putative coupling site of the reductasethatsupplieselectronsfor aromatization. The siteis larger than the active site, and at least twice aslarge asother P450s.MutantsK440Qand Y361Fof this region are virtuallyinactive.Collectivelythe results suggestfunctional significanceof oligomerization. Several newly designedAIs are superiortoexemestane, the steroidal AI currently used as a drug, in inhibition and anti-proliferation assays. The C6β-(pent-2-yn-1-yloxy) side chains ofthe most potent compoundspenetrate the access channelunique to AROM and havethe sameconformation asin the enzyme-free state.Astructural-based approachcan improve drug efficacy by improving specificity and selectivity, and reducing sideeffects.
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