Bacterial L-asparaginases, enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Other substrates of asparaginases include L-glutamine, D-asparagine, and succinic acid monoamide. In this report, we present high-resolution crystal structures of the complexes of Erwinia chrysanthemi L-asparaginase (ErA) with the products of such reactions that also can serve as substrates, namely L-glutamic acid (L-Glu), D-aspartic acid (D-Asp), and succinic acid (Suc). Comparison of the four independent active sites within each complex indicates unique and specific binding of the ligand molecules; the mode of binding is also similar between complexes. The lack of the alpha-NH3(+) group in Suc, compared to L-Asp, does not affect the binding mode. The side chain of L-Glu, larger than that of L-Asp, causes several structural distortions in the ErA active side. The active site flexible loop (residues 15-33) does not exhibit stable conformation, resulting in suboptimal orientation of the nucleophile, Thr15. Additionally, the delta-COO(-) plane of L-Glu is approximately perpendicular to the plane of gamma-COO(-) in L-Asp bound to the asparaginase active site. Binding of D-Asp to the ErA active site is very distinctive compared to the other ligands, suggesting that the low activity of ErA against D-Asp could be mainly attributed to the low k(cat) value. A comparison of the amino acid sequence and the crystal structure of ErA with those of other bacterial L-asparaginases shows that the presence of two active-site residues, Glu63(ErA) and Ser254(ErA), may correlate with significant glutaminase activity, while their substitution by Gln and Asn, respectively, may lead to minimal L-glutaminase activity.

4,7-Dioxosebacic acid (4,7-DOSA) is an active site-directed irreversible inhibitor of porphobilinogen synthase (PBGS). PBGS catalyzes the first common step in the biosynthesis of the tetrapyrrole cofactors such as heme, vitamin B 12 , and chlorophyll. 4,7-DOSA was designed as an analogue of a proposed reaction intermediate in the physiological PBGS-catalyzed condensation of two molecules of 5-aminolevulinic acid. As shown here, 4,7-DOSA exhibits time-dependent and dramatic species-specific inhibition of PBGS enzymes. IC 50 values vary from 1 µM to 2.4 mM for human, Escherichia coli, Bradyrhizobium japonicum, Pseudomonas aeruginosa, and pea enzymes. Those PBGS utilizing a catalytic Zn 2+ are more sensitive to 4,7-DOSA than those that do not. Weak inhibition of a human mutant PBGS establishes that the inactivation by 4,7-DOSA requires formation of a Schiff base to a lysine that normally forms a Schiff base intermediate to one substrate molecule. A 1.9 Å resolution crystal structure of E. coli PBGS complexed with 4,7-DOSA (PDB code 1I8J) shows one dimer per asymmetric unit and reveals that the inhibitor forms two Schiff base linkages with each monomer, one to the normal Schiff base-forming Lys-246 and the other to a universally conserved "perturbing" Lys-194 (E. coli numbering). This is the first structure to show inhibitor binding at the second of two substrate-binding sites.Porphobilinogen synthase (PBGS, 1 EC 4.2.1.24, also known as 5-aminolevulinic acid dehydratase) is a highly conserved metalloenzyme that functions in the first common step of the biosynthesis of the essential tetrapyrroles. The PBGS-catalyzed reaction is an asymmetric condensation between two molecules of 5-aminolevulinic acid (ALA) as described in Figure 1A. The crystal structure of the enzyme has been established for PBGS from yeast, Escherichia coli, and Pseudomonas aeruginosa (1-3). Some of the published structures contain the bound inhibitor levulinic acid, a complex analogous to the first enzyme-bound intermediate (2-4), which is a Schiff base formed between the keto group of P-side ALA (see Figure 1A) and the amino group of an invariant lysine residue (see Figure 2A). These structures delineated the locations of several different divalent metal ions and the binding residues for the carboxylic acid moieties of the two ALA substrate molecules. Despite the fact that PBGS is an octamer of ∼300 kDa, the catalytic and binding residues at each active site derive from only one subunit. † This work was supported by Grant ES03654 (E.K.J.) from the National Institute of Environmental Health Sciences, NIH, by NIH Grant CA06927 (ICR), and by an appropriation from the Commonwealth of Pennsylvania. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute nor of the National Institute of Environmental Health Sciences.* To whom correspondence and reprint requests should be addressed. Telephone: 215-728-3695. Fax: 215-728-2412. E-mail: EK_Jaffe@fccc.edu.‡ Fox ...

The X-ray structure of ribonuclease-A refined in London and the joint X-ray and neutron structure refined in Washington are compared. The two structures are shown to be very similar, the mean difference between the protein atoms of the two structures being 0.374 A, The two models differ in assignments of atomic positions of the active-site side-chain atoms in Lys 41 and His 119 and these differences may be due to different conformations in the two structures. Strongly bound solvent occupies the same position in both structures but there is some evidence of other solvent structure being different, possibly owing to the presence of different alcohols or the use of partial deuteration in Washington. Temperature factors in both structures show similar trends. 0108-7681/86/040379-09501.50 O 1986 International Union of Crystallography

The QM/MM MD and free energy simulations show that serine-carboxyl peptidases (sedolisins) may stabilize the tetrahedral intermediates and tetrahedral adducts primarily through a general acid-base mechanism involving Asp (Asp164 for kumamolisin-As) rather than the oxyanion-hole interactions as in the cases of serine proteases.