Enzyme crystal structure in a neat organic solvent.

Abstract

The crystal structure of the serine protease subtllsln Carbsberg In anhydrous acetonitrile was determined at 2.3 A rolution. It was found to be easentially Identical to the tbree-dimensional structure of the enzyme In water; the differences observed were smaller than those between two lndependentiy determined struchre In aqueous solution. The hydrogen bond system of the catalytic triad is intact in acetonitrile. The majority (99 of 119) of enzyme-bound, structural water molcules have such a great affnity to subillsin that they are not dipld even in anhydrous acetonltrile. Of the 12 enzyme-bound acetonitrile mdsoules, 4 dispiace water molecules and 8 bind where no water had been observed before. One-third of all subtin-bound acetonitrile molecules reside in the active center, occupying the same region (PI, P2, and P3 bnding dtes) as the specfic protein Inhibitor eglin c.The past several years have witnessed the emergence of nonaqueous enzymology (1-5); it has become clear that not only can ordinary enzymes [and antibodies (6-8)] function in organic solvents containing little or no water, but when placed in such an utterly unnatural milieu they exhibit unusual properties, including the ability to catalyze additional reactions, markedly altered specificity, and "molecular memory." To understand the mechanistic basis of these phenomena, it is critical to elucidate protein conformation in organic media and compare it with that in aqueous solution.This task has been achieved in the present work by means of high-resolution x-ray crystallography; remarkably, the threedimensional structure of the serine protease subtilisin Carlsberg in anhydrous acetonitrile has been found to be essentially indistinguishable from that in water. § Moreover, much of the water structure around the protein is identical to that in aqueous medium. In addition, several acetonitrile molecules are uniquely bound to the protein, mapping an interaction surface distinct from that for water. METHODS Cyt. Subtilisin Carlsberg, an alkaline serine protease from Bacillus licheniformis (EC 3.4.21.14) purchased from Sigma (type VIII), was crystallized by batch precipitationtfrom aqueous Na2SO4 as described (9). The long prismatic crystals obtained were divided into two portions. The first was used to determine the enzyme structure in water at a higher resolution than is currently available (10), so that the number and positions of bound water molecules could be established. The second batch of crystals was used for nonaqueous structure analysis. Solvent Inion. Attempts to gradually replace the interstitial water with acetonitrile failed; when the organic solvent content reached aw50%6 (vol/vol), the enzyme crystals invariably fractured. To fortify the crystals, we cross-linked them lightly with glutaraldehyde following the general methodology of . [Cross-linked enzyme

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

“…Previously, we solved the x-ray crystal structures of lightly crosslinked subtilisin Carlsberg in dioxane (17), acetonitrile (15), and water (16) and found them virtually identical, with an rmsd of the backbone atoms of 0.3 Å among the different structures. However, to determine whether the enzyme mechanism is the same in organic solvent as in water, one must examine the structure of a reaction intermediate formed in both media, not just the structure of the free enzyme.…”

“…Rigid body refinement (10-4.0 Å) was performed using X-PLOR (29) for acetonitrile and water data and coordinates for the corresponding cross-linked nonacylated crystal structure PDB 1scb and 1sca (15,16).…”

“…A total of 62 water molecules were included in the model in acetonitrile and 88 water molecules and 1 Ca 2ϩ molecule in the model in water. Organic solvent molecules similarly were introduced into the model as appropriate using the acetonitrile model (15), and parameter and topology files generated with the aid of XPLO2D (33). Acetonitrile electron density was recognized by comparison with F calc maps calculated for a theoretical acetonitrile molecule between the resolution limits of 14-2.15 Å.…”

“…Such an understanding, like that concerning the selectivity, requires both structural and mechanistic information. Recently, structures of several enzymes in neat organic solvents, namely those of subtilisin Carlsberg in acetonitrile (15,16) and dioxane (17), ␥-chymotrypsin in hexane (18,19), and elastase in acetonitrile (20), have been elucidated and found to be essentially the same as in water. Furthermore, the structures in hexane of a ␥-chymotrypsin-product complex has been determined (19) and that of a tetrahedral complex claimed (18).…”

New insights into the molecular mechanism of methanol-induced inactivation ofThermomyces lanuginosuslipase: a molecular dynamics simulation study

“…The result obtained from MD simulation of WT TLL in methanol is consistent with a previous circular dichroism spectra study of WT TLL in high concentration isopropanol solutions (up to 50%), which revealed that isopropanol molecules did not cause major alterations in secondary structure, although the enzyme became completely inactive in 50% isopropanol [34]. X-ray structures of serine proteases, subtilisin Carlsberg and porcine pancreatic elastase, crystallized in neat acetonitrile, 80% (v/v) isopropanol and 80% (v/v) ethanol, respectively, also confirmed a native-like secondary structure of each enzyme [35] and [14].…”