The crystal structure of Escherchia coli asparaginase II (EC 3.5.1.1), a drug (Elspar) used for the treatment of acute lymphoblastic leukemia, has been determined at 2.3 A resolution by using data from a single heavy atom derivative in combination with molecular replacement. The atomic model was refined to an R factor of 0.143. This enzyme, active as a homotetramer with 222 symmetry, belongs to the class of a/P proteins. Each subunit has two domains with unique topological features. On the basis of present structural evidence consistent with previous biochemical studies, we propose locations for the active sites between the N-and C-terminal domains belonging to different subunits and postulate a catalytic role for Thr-89.

The structure of a crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site has been determined. The sequence of the inhibitor JG-365 is Ac-Ser-Leu-Asn-Phe-#[CH(OH)CH2N]-Pro-Ile-ValOMe; the K; is 0.24 nM. The hydroxyethylamine moiety, in place of the normal scissile bond of the substrate, is believed to mimic a tetrahedral reaction intermediate. The structure of the complex has been rermed to an R factor of 0.146 at 2.4A resolution by using restrained least squares with rms deviations in bond lengths of 0.02 A and bond angles of 4'. The bound inhibitor diastereomer has the S configuration at the hydroxyethylamine chiral carbon, and the hydroxyl group is positioned between the active site aspartate carboxyl groups within hydrogen bonding distance. Comparison of this structure with a reduced peptide bond inhibitor-protease complex indicates that these contacts confer the exceptional binding strength of JG-365.Reverse transcriptase, integrase, and protease are the three virally encoded enzymes necessary for replication of human immunodeficiency virus 1 (HIV-1), so each is a potential target for drug design. Rational design of drugs directed against AIDS would be greatly facilitated by knowledge of the three-dimensional structures of the target molecules, yet the protease is the only one of these enzymes for which the structure of the native form (1-3) or of an inhibitor complex (4) is known. The protease is a member of the wellcharacterized family of aspartic proteases, which also includes mammalian enzymes such as renin, pepsin, and chymosin. Whereas cell-encoded aspartic proteases are monomers with distinct amino and carboxyl domains, the retroviral proteases are dimers of identical subunits that are analogous to these domains (5). The function of the HIV-1 protease is to cleave the translated viral gag-pol polyprotein into discrete components. Without protease activity, the viral particle remains noninfective (6, 7) and this property makes the protease an attractive candidate for therapeutic drug design against AIDS (8)(9)(10)(11)(12)(13)(14).Inhibitors -of aspartic proteases have been developed as potential pharmaceutical agents for modulating the biological processes catalyzed by this class of enzymes (15,16 Herein we report the structure of a synthetic HIV-1 protease complexed with an HEA inhibitor, JG-365, and compare it with a complex structure in which the reduced peptide bond inhibitor MVT-101 was 3000 times less potent (4). The results substantiate a previously proposed mechanism of action for this class of enzymes (23) protease (where Aba is L-a-amino-n-butyric acid) was chemically synthesized as previously described (24,25). The sequence used was that of the SF2 isolate with cysteines replaced by L-a-amino-n-butyric acid (2). The inhibitor JG-365 was synthesized as described (22) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby...

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