Biochemistry zyxwvut 1995, zyxwvut 34, zyxwvu 11515 zyxwv - 11520 11515 Glutamate zyxwvu y -Semialdehyde as a Natural Transition State Analogue Inhibitor of Escherichia coli Glucosamine-6-phosphate Synthase? Stephen L. Bearne and Richard Wolfenden* Department of Biochemistry and Biophysics, University zyxwvu of North Carolina, Chapel Hill, North Carolina 27599-7260 Received April 4, 1995; Revised Manuscript Received June 12, 1995@ ABSTRACT: Pyrroline-5-carboxylate, an intermediate in the biosynthesis and degradation of glutamate, proline, and ornithine, acts as a strong reversible inhibitor of glucosamine-6-phosphate synthase, competitive with respect to glutamine. Proton magnetic resonance spectroscopy shows that, under these conditions, pyrroline-5-carboxylate exists in rapid equilibrium with glutamate y-semialdehyde (0.05%). The observed variation of Ki with pH is consistent with inhibition by this rare species. Glutamate y-semialdehyde is expected to react reversibly with a cysteine residue at the active site, identified by earlier inactivation studies. to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. The apparent Ki value of glutamate y-semialde6yde is approximately 3 x LO-* M. Glucosamine-6-phosphate synthase (L-g1utamine:D-fructose- 6-phosphate amidotransferase; GlmS;' E.C. 2.6.1.16) cata- lyzes the first step in hexosamine metabolism, converting fructose-6-phosphate (Fru-6-P) into glucosamine-6-phosphate using glutamine as the ammonia source (Scheme 1) (Gosh et al., 1960; Badet-Denisot et al., 1993). Glucosamine-6- phosphate is eventually transformed into uridine diphospho- N-acetylglucosamine, from which other amino sugar- containing molecules are derived. One of these products, N-acetylglucosamine, is a major constituent of the pepti- doglycan layer of bacterial and fungal cell walls. Accord- ingly, GlmS offers a potential target for antibacterial and antifungal agents (Andruszkiewicz et al., 1990; Badet- Denisot et al., 1993). In the sequence of reactions catalyzed by glucosamine- 6-phosphate synthase, glutamine is first hydrolyzed to yield glutamate and nascent ammonia, and ammonia is then transferred to fructose-6-phosphate. Finally, Fru-6-P is isomerized from a ketose to an aldose, in a reaction corresponding to a Heyns rearrangement (Kort, 1970; Golinelli-Pimpaneau et al., 1989). GlmS is inactivated by iodoacetamide (Badet et al., 1987), the glutamine analogue 6-diazo-5-oxonorleucine (Gosh et al., 1960; Badet et al., 1987), and #-fumaroyl-~-2,3-diaminopropionate derivatives (Kucharczyk et al., 1990). Each of these reagents derivatizes the thiol function of the N-terminal cysteine residue, which is believed to form a y-glutamyl thioester intermediate during glutamine hydrolysis (Scheme 2A). In accord with this scheme, conversion of the N-terminal cysteine residue to alanine by site-directed mutagenesis results in a total loss of enzymatic activity (Badet-Denisot et al., 1993). Aldehydes, structurally related to the acyl portion of substrates, act as potent reversible inhibitors of proteases that + This work was supported by NIH Grant GM-18325. S.L.B. is grateful to the Natural Sciences and Engineering Research Council of Canada for support through a postdoctoral fellowship. * Corresponding author. @ Abstract published in Advance ACS Abstracts, August 15, 1995. I Abbreviations: DSS, 3-(trimethylsilyl)propanesulfonic acid; Fru- 6-P, fructose-6-phosphate; GlmS, glucosamine-6-phosphate synthase; Gln, glutamine; GSA, glutaric semialdehyde; P2C, Al-pyrroline-2- carboxylate; P5C, A'-pyrroline-5-carboxylate. contain a reactive cysteine (Westerik & Wolfenden, 1972) or serine (Thompson, 1973) residue at the active site. Inhibition of papain involves formation of a thiohemiacetal (Lewis & Wolfenden, 1977; Bendall et al., 1977; Mackenzie et al., 1986) that appears to resemble intermediates in the formation and breakdown of a covalently bound thiol ester intermediate in substrate hydrolysis. If the cysteine residue at the active site of GlmS were to perform a similar function in glutamine cleavage, we reasoned that glutamate y-semi- aldehyde might serve as a strong reversible inhibitor (Scheme 2B). Of special interest is the fact that glutamate y-semi- aldehyde exists in unfavorable equilibrium with pyrroline- 5-carboxylate (P5C) (Schopf & Oechler, 1936; Schopf & Steuer, 1947; Vogel & Davis, 1952; Mezl & Knox, 1976), an intermediate in the metabolism of glutamate, proline, and ornithine [for reviews, see Jones (1983, 1985)l. This paper describes the inhibition of Escherichia coli glucosamine-6- phosphate synthase by glutamate y-semialdehyde and the position of its equilibrium of cyclization to form P5C. MATERIALS AND METHODS Q-Sepharose Fast Flow and Phenyl Sepharose CL-4B chromatography gels were purchased from Pharmacia, Inc. Ultrogel AcA 34 was purchased from Sepracor Corp. A1-Pyrroline-5-carboxylate (P5C). P5C was synthesized by oxidation of a mixture of D,L- and D,L-allo-&hydroxy- lysine with periodic acid and purified by ion-exchange chromatography as described by Williams and Frank (1975). The concentration of P5C was determined by the colorimetric method of Mezl and Knox (1976), using the molar extinction coefficient (E) of the condensation product of P5C with o-aminobenzaldehyde (E = 2580 M-' cm-') reported by these authors. A1-Pyrroline-2-carboxylate (P2C). P2C was synthesized by treatment of 3,3-dichloro-2-piperidone with barium hydroxide as described by Lewis et al. (1993) and Osugi (1958). 3,3-Dichloro-2-piperidone was prepared by treat- ment of 2-piperidone with PCls and chlorine as described by Wineman et al. (1958). Glutaric Semialdehyde. Glutaric semialdehyde (GSA) was prepared by oxidation of D,L-a-aminoadipic acid with 0006-2960/95/0434- 11515$09.00/0 0 1995 American Chemical Society