Trypanothione reductase from Crithidia fasciculata has been purified ca. 1400-fold to homogeneity in an overall yield of 60%. The pure enzyme showed a pH optimum of 7.5-8.0 and was highly specific for its physiological substrates NADPH and trypanothione that had Km values of 7 and 53 microM, respectively. Trypanothione reductase was found to be a dimer of identical subunits with Mr 53 800 each. The enzyme displayed a visible absorption spectrum that was indicative of a flavoprotein with a lambda max at 464 nm. The flavin was liberated by thermal denaturation of the protein and identified, both by high-performance liquid chromatography (HPLC) and by fluorescence studies, as FAD. The extinction coefficient of pure enzyme at 464 nm was determined to be 11.3 mM-1 cm-1. Upon titration with 5,5'-dithiobis(2-nitrobenzoic acid), oxidized enzyme was found to contain 2.2 (+/- 0.1) free thiols, whereas NADPH-reduced enzyme showed 3.9 (+/- 0.3). Furthermore, whereas oxidized enzyme was stable toward inactivating alkylation by 2.0 mM iodoacetamide, NADPH-reduced enzyme was inactivated with a half-life of 14 min. These data suggested that a redox-active cystine residue was present at the enzyme active site. Upon reduction of the enzyme with 2 electron equiv of dithionite, a new peak in the absorption spectrum was observed at 530 nm, thus indicating that a charge-transfer complex between one of the newly reduced thiols and the oxidized FAD had formed.(ABSTRACT TRUNCATED AT 250 WORDS)

To probe the catalytic mechanism of human purine nucleoside phosphorylase (PNP), 13 active-site mutants were constructed and characterized by steady-state kinetics. In addition, microtiter plate assays were developed for both the phosphorolytic and synthetic reactions and used to determine the kinetic parameters of each mutant. Mutations in the purine binding site exhibited the largest effects on enzymatic activity with the Asn243Ala mutant resulting in a 1000-fold decrease in the kcat for inosine phosphorolysis. This result in combination with the crystallographic location of the Asn243 side chain suggested a potential transition state (TS) structure involving hydrogen bond donation by the carboxamido group of Asn243 to N7 of the purine base. Analogous to the oxyanion hole of serine proteases, this hydrogen bond was predicted to aid catalysis by preferentially stabilizing the TS as a consequence of the increase in negative charge on N7 that occurs during glycosidic bond cleavage and the associated increase in the N7-Asn243 hydrogen bond strength. Two residues in the phosphate binding site, namely His86 and Glu89, were also predicted to be catalytically important based on their alignment with phosphate in the X-ray structure and the 10-25-fold reduction in catalytic activity for the His86Ala and Glu89Ala mutants. In contrast, catalytic efficiencies for the Tyr88Phe and Lys244Ala mutants were comparable with wild-type, indicating that the hydrogen bonds predicted in the initial X-ray structure of PNP [Ealick, S. E., et al. (1990) J. Biol. Chem. 265, 1812-1820] were not essential for catalysis. These results provided the foundation for studies reported in the ensuing two manuscripts focused on the PNP catalytic mechanism [Erion, M. D., et al. (1997) Biochemistry 36, 11735-11748] and the use of mutagenesis to reverse the PNP substrate specificity from 6-oxopurines to 6-aminopurines [Stoeckler, J. D., et al. (1997) Biochemistry 36, 11749-11756].

Carbon-phosphorus bond cleavage activity, found in bacteria that utilize alkyl-and phenylphosphonic acids,has not yet been obtained in a cell-free system. Given this constraint, a systematic examination of in vivo C-P lyase activity has been conducted to develop insight into the C-P cleavage reaction. Six bacterial strains were obtained by enrichment culture, ideptified, and characterized with respect to their phosphonic acid substrate specificity. One isolate, Agrob4acterium radiobacter, was shown to cleave the carbon-phosphorus bond of a wide range of substrates, including fosfomycin, glyphosate, and dialkyl phosphinic acids. Furthermore, this organism processed vinyl-, propenyl-, and prQpynylphosphonic acids, a previously uninvestigated group, to ethylene, propene, and propyne, respectively. A determination of product stoichiometries revealed that both C-P bonds of dimethylphosphinic acid are cleaved quantitatively to methane and, furthermore, that the extent of C-P bond cleavage correlated linearly with the specific growth rate for a range of substrates. The broad substrate specificity of Agrobacterium C-P lyase and the comprehensive characterization of the in vivo activity make this an attractive system for further biochemical and mechanistic experiments. In addition, the failure to observe the activity in a group of gram-positive bacteria holds open the possibility that a periplasmic component may be required for in vivo expression of C-P lyase activity.Living systems contain organophosphorus mostly in the form of oxygen esters, diesters, and anhydrides of phosphoric acid. However, phosphonic acids, a class of organophosphorus compounds containing a direct carbon-phosphorus bond, are also found in nature (14, 21). The initial discovery of a natural phosphonic acid product, 2-aminoethylphosphonic acid, in 1959 (18) is relatively recent compared with the history of research in biological organophosphate esters. Since that time dozens of naturally occurring C-P compounds have been discovered (17). The biosynthesis of the C-P bond has been documented in protozoa, fungi, and molluscs (16, 37). The importance of C-P bonds in these invertebrates is underscored by the observation that 50 to 75% of the cilia phospholipids of Tetrahymena are phosphonolipids (22).Given the biosynthetic accessibility of the C-P linkage it is almost axiomatic that bacteria would have evolved the ability to catabolize phosphonic acids. Indeed, by enrichment culture bacteria have been obtained that utilize 2-aminoethylphosphonic acid, the most abundant naturally occurring phosphonic acid, as the sole source of phosphorus, nitrogen, and carbon (4). The biochemistry of this process was first investigated in Bacillus cereus, where 2-aminoethylphosphonic acid was shown to undergo transamination to yield phosphonoacetaldehyde followed by hydrolysis to acetaldehyde and Pi (25). The hydrolytic cleavage step is catalyzed by phosphonoacetaldehyde hydrolyase (26), an enzyme given the trivial name of phosphonatase (equation 1).2-phosphonatase OHCCH...

A mixture of ananain (EC 3.4.22.31) and comosain purified from crude pineapple stem extract was found to contain numerous closely related enzyme forms. Chromatographic separation of the major enzyme forms was achieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroacetone or iodoacetamide gave enzyme for structural analyses by 19F-n.m.r. and electrospray mass spectrometry respectively. Structural and kinetic analyses revealed comosain to be closely related to stem bromelain (EC 3.4.22.32), whereas ananain differed markedly from both comosain and stem bromelain. Nevertheless, differences were seen between comosain and stem bromelain in amino acid composition and kinetic specificity towards the epoxide inhibitor E-64. Differences between five isolatable alternative forms of ananain were characterized by amidolytic activity, thiol stoichiometry and accurate mass determinations. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid.