Using N-acetylglucosaminono-I ,5-lactone (1) as the reference, the inhibitory activity of its (formal) derivatives N-acetylglucosaminono-1,5-lactone oxime (2) and N-acetylglucosaminono-1,5-lactone 0-(phenylcarbamoy1)-oxime (3) was tested against P-N-acetylglucosaminidase of different origins (animal, plant, fungus). Displaying inhibition constants of 0.45 pM and 0.62 pM, for the animal and plant enzyme, respectively, the simple oxime 2 was about equally potent as the parent lactone 1, and 50-400 times more efficient than two recently described new P-N-acetylglucosaminidase inhibitors. The (phenylcarbamoy1)oxime 3 performed even better, particularly with the fungal enzyme (Ki = 40 nM), i.e. was about 350 times more potent than the lactone. In all cases competitive inhibition was observed with 4-nitrophenyl-~-N-acetylglucosaminide as the substrate. With K J K , ratios up to 3300 for 2 and 13600 for 3, the mode of action of these novel inhibitors is probably that of transition state mimicry. Suggestions are made for their use as a tool in biological research.Aldono-l,5-lactones are well known potent competitive inhibitors of glycosidases acting on substrates of the corresponding configuration [l, 21. In particular, this relates to the inhibition of P-N-acetylglucosaminidase (P-GlcNAc'ase; EC 3.2.1.30) by N-acetylglucosaminono-1,5-lactone (1; see Fig. 1) [3], although with values ranging over 0.8-660 pM [4-91 inhibitor constants display considerable variation. Such lactones are thought to adopt a conformation similar to that of a glycopyranosyl cation and to be effective as transitionstate approximates [lo] (for current views of the theory of transition state affinity and the design of enzyme inhibitors, see 11 1, 121).As glucono-l,5-lactone oxime and its phenylcarbamoyl derivative were found to be about equal or even better inhibitors of P-glucosidase than the parent lactone [ 131, investigations into the effect of the corresponding N-acetylglucosamine derivatives 2 and 3 on p-GlcNAc'ase seemed promising. In addition, to estimate the extent to which the source of the P-GlcNAc'ase might have a bearing on its susceptibility to inhibition, experiments were performed with enzymes of widely different organisms. A preliminary account of part of this work has been published [14]. Abbreviations. 4-Np-GlcNAc, 4-nitrophenyl-~-N-acetylglucosaminide; P-GlcNAc'ase, P-N-acetylglucosaminidase. MATERIALS AND METHODS SourcesEnzyme. P-N-Acetylglucosaminidase (EC 3.2.1.30).Chemical Co.), but the enzyme preparation from Mucor rouxii was obtained in this laboratory as follows. Log-phase hyphae of the fungus were cultivated [15] and the cells washed thoroughly with 20 mM Bistris pH 6.5, drained, and disrupted with glass beads (0.5 mm) in an MSK homogenizer (Braun, Melsungen) for 60 s at 4000 rpm. After removal of the beads, followed by centrifugation at 4000 x g for 5 min, and then at 54 500 x g for 45 min, the supernatant ( z 70 ml) was subjected to anion-exchange chromatography, using a column of DEAE-Sephadex A-25 (d 50 x 150 mm) and...

Enzymological evidence has been sought for the purported involvement of chitinolysis in vegetative growth of filamentous fungi. A procedure has been developed for the production of fast growing and morphologically homogeneous exponential phase mycelium of the non-septate dimorphic zygomycete Mucor rouxii. A partially purified extract of this material has been subjected to gel-permeation chromatography and the chitinolytic activity of eluate fractions has been assessed using colloidal and nascent chitin and 3,4-dinitrophenyl tetra-Nacetylchitotetraoside [3,4-DNP-(GlcNAc),] as substrates. Exponentially growing (fa = 1.1 h) mycelium consisting of single short-branched hyphae contains at least seven chitinases. The two particulate ones have not been studied in detail. The soluble chitinases hydrolyse (pseudo)chito-oligomers by random cleavage of internal p-1,4-bonds (and not by processing) and have a minimum chain-length requirement of n = 4. They are clearly distinct from p-N-acetylglucosaminidase (P-GlcNAc'ase) with respect to their chromatographic behaviour, substrate chain-length specificity, inhibition by chitobionolactone oxime (Ki = 175 MI, and non-inhibition by the specific PGlcNAc'ase inhibitor N-acetylglucosaminono-1,5-lactone oxime. Their pH optima are similar (6.!5-7.0), and all can hydrolyse 3,4-DNP-(GlcNAc), as well as nascent chitin. With respect to their charge, response to protease treatment, behaviour upon gel-permeation chromatography and ability to use colloidal chitin as a substrate, the soluble chitinases do, however, represent two distinct groups. Type A chitinases are acidic, display partial latency, show an unusual affinity to dextran gel and act weakly on colloidal chitin. Type B chitinases are basic (or neutral) and non-zymogenic, do not behave anomalously upon gel filtration and can degrade preformed chitin. An hypothesis is presented for the function of the complex chitinolytic system of the fungal hypha in branching and, possibly, also in apical growth.

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The substrate kinetics of chitin synthase (CS) were non-Michaelian, irrespective of the type of enzyme preparation studied ( 1 0 5 s chitosomal CS, and 16-S CS ex walls), even in the presence of saturating GlcNAc. An unexplained idiosyncrasy of this enzyme, which is likely to be responsible for this phenomenon, is evident from the striking non-linearity of product deposition with time at low substrate or low enzyme concentrations, particularly in the absence of GlcNAc. The possibility can be excluded that this non-linearity is due to the formation of soluble by-products or intermediates in the form of chito-oligomers, as shown by HPLC/pulsed amperometric detection analysis. Additional evidence was sought for the tenet that CS is homotropicall y-heterotropically regulated, at least under steady-state reaction conditions. Substrate kinetic curves established from rate data for the linear reaction phase only were used for modelling. These could be reasonably well parameterised on the basis of the Monod mathematical model for co-operative ligand binding. Within a series of test compounds used to assess the stereochemical conditions of the allosteric site of CS for effector binding, N-acetylglucosaminono-I ,5-lactone oxime excelled. Requirements for effector binding are as follows: (a) an aminoglucopyranose skeleton with the amino function acetylated, and (b) a single-bonded 0x0-function present at C(1), which is preferentially a hydrogen bond donor, that may be equatorially spaced off, but must not be a-anomeric. The implications of these findings for chitin synthesis in vivo are discussed in terms of a mechanistically based fitness of CS to operate efficiently under vastly different combinations of substrate and effector concentrations as well as with respect to the conditions under which chitin synthase could be coordinately linked to the catabolism of chitin.