Resonance Raman Spectroscopy of the Oxygenated Intermediates of Human CYP19A1 Implicates a Compound I Intermediate in the Final Lyase Step

CYP19A1, or aromatase, a cytochrome P450 responsible for estrogen biosynthesis in humans, is an important therapeutic target for the treatment of breast cancer. There is still controversy surrounding the identity of reaction intermediate that catalyzes carbon–carbon scission in this key enzyme. Probing the oxy-complexes of CYP19A1 poised for hydroxylase and lyase chemistries using resonance Raman spectroscopy and drawing a comparison with CYP17A1, we have found no significant difference in the frequencies or isotopic shifts for these two steps in CYP19A1. Our experiments implicate the involvement of Compound I in the terminal lyase step of CYP19A1 catalysis

Spectroscopic characterization of iron-oxygen intermediates in human aromatase (CYP19A1)

CYP19A1 or aromatase, is a human steroidogenic P450 important for estrogen biosynthesis in humans. Over activation of aromatase results in malignancies of the breast tissue, especially in post menopausal women. In fact, aromatase inhibitors constitute the front line therapy for estrogen receptor positive (ER+) breast cancer in post-menopausal women which accounts for over 70% of all breast cancer cases in the United States. Starting with its androgenic substrates, testosterone and androstenedione, CYP19A1 forms estradiol and estrone utilizing one molecule of atmospheric oxygen and two reducing equivalents in the form of NADPH. This is accomplished in a three-step process one of which involves a carbon-carbon bond scission and aromatization. The catalytic mechanism of P450s has been long studied and it is well known that an oxo-ferryl π-cation radical, known as “Compound 1” in P450 chemistry is the reactive intermediate that catalyzes most of the reactions of P450s. The identity of the reaction intermediate that catalyzes the terminal step estrogen biosynthesis by CYP19A1 is still a mystery. There is evidence in the literature suggesting the involvement of Compound 1 via a hydrogen abstraction that initiates deformylation and subsequent aromatization. There is also suggestion of the peroxo-anion or “Compound 0” acting as a nucleophile, attacking the electrophilic carbonyl carbon of 19-oxo-androstenedione forming a peroxide adduct that then fragments to produce acyl-carbon cleavage. Owing to the interesting chemistry CYP19A1 catalyzes and its role in human health I focused my attention towards elucidating the mechanism of this critical enzyme with the hope that a detailed picture of the workings of CYP19A1 will help guide efforts to make more specific inhibitors and improve breast cancer prognosis. CYP19A1 is a membrane-bound hemeprotein with a rich spectroscopic landscape thus affording an opportunity to apply a repertoire of biophysical approaches to help piece together a reaction mechanism. I used the Nanodisc technology to stabilize CYP19A1 in its native membrane-like environment to obtain a mono-disperse, stable and homogenous enzyme preparation that is amicable to the optical, resonance Raman (rR) and electron paramagnetic resonance (EPR) spectroscopy and also, cryoradiolysis and cryospectroscopy. The approach I have applied in this project has been that of characterizing the individual fate of reaction intermediates on their way from substrates to products thereby catching them ‘in action’. My cryospectroscopy, EPR, rR and steady state kinetics efforts outlined in this doctoral thesis all implicate “Compound 1” as the reactive intermediate that is responsible for the carbon-carbon scission reactivity of CYP19A1

A method for computing the inter-residue interaction potentials for reduced amino acid alphabet

Inter-residue potentials are extensively used in the design and evaluation of protein structures. However, dealing with all (20×20) interactions becomes computationally diffi cult in extensive investigations. Hence, it is desirable to reduce the alphabet of 20 amino acids to a smaller number. Currently, several methods of reducing the residue types exist; however a critical assessment of these methods is not available. Towards this goal, here we review and evaluate different methods by comparing with the complete (20×20) matrix of Miyazawa-Jernigan potential, including a method of grouping adopted by us, based on multi dimensional scaling (MDS). The second goal of this paper is the computation of inter-residue interaction energies for the reduced amino acid alphabet, which has not been explicitly addressed in the literature until now. By using a least squares technique, we present a systematic method of obtaining the interaction energy values for any type of grouping scheme that reduces the amino acid alphabet. This can be valuable in designing the protein structures