Abstract
Crystals of the steroid-metabolizing enzyme, delta 5-3-ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni, exhibit many enzymatic properties. Each enzyme subunit in the lattice binds a competitive inhibitor, progesterone, with the same stoichiometry (1:1) and affinity (KD = 6 X 10(-6) M) as the enzyme in solution. Another competitive inhibitor, 19-nortestosterone, competes with progesterone for the same binding sites in the crystal. The enzyme crystals catalyze the conversion of delta 5- to delta 4-ketosteroids, but because the enzyme is so efficient, and substrate diffusion into the crystal is so slow, substrate cannot penetrate deeply into the crystal before being converted to product. A general theoretical formulation is presented to account for the effects of substrate diffusion into enzyme crystals of different shapes and sizes. The dependence of apparent mean enzyme activity in steroid isomerase crystals as a function of crystal size is shown to be consistent with this theoretical formulation. These inhibitor binding and catalytic properties suggest that the enzyme is in an active conformation within these crystals.
Highlights
Crystals of the steroid-metabolizing enzyme, A8-3- For a typical substrate, hexagonal crystals of the A5-3-ketoketosteroid isomerase (EC 5.3.3.1) from Pseudomonas steroid isomerase [15] have an estimated diffusion length that testosteroni, exhibit many enzymatic properties
These in- function of crystal size and fit these measurements to theohibitor binding and catalytic properties suggest that retical predictions that account for diffusion limitations
R = - 3 s a a 1 p E tanh(pa) pa Insolubility of A5-3-Ketosteroid Isomerase Crystals in Stabilizer-The assay for A5-3-ketosteroid isomerase is capable of detecting 0.12 pmol/ml of enzyme [16], but no enzyme activity was detected in supernatant stabilizer solution
Summary
(reaction velocity for dissolved A'-3-ketosteroid isomerase) one in 17 pl of methanol and 24.5 nmol of labeled glycine in 10 pl of water were added to 173p1 of stabilizer solution in precisely the same. = volume of the Isomerase Crystals-As described by Penning et al [18], 10 nmol of [3H]progesterone (1.05 X lo cpm/nmol) in 10 /rl of methanol were added to 100pl of stabilizer solution. The remaining solution was subjected to quantitative amino acid [ZE]=concentration at equilibrium of inhibitor-enzyme complex within the crystal. Michaelis-Menten kinetics require that the steady state rate of conversion of substrate toproduct be: rate of product formationper enzyme molecule in the crystal: : s” ‘OD‘ R = a2Ds0 2pE cosh(ap) o cosh(ar - ap)dr = - tanh(ap). Carboxypeptidase crystals have an estimateddiffusion length of 6 pm [9]
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