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Kinetic data for the 6'-aminoglycoside-modifying enzyme, AAC(6')-4, also named kanamycin acetyltransferase, have been collected for six aminoglycoside antibiotics (amikacin, gentamicin C1a, kanamycin A, neomycin B, sisomicin, and tobramycin) and three coenzymes (acetyl-CoA, n-propionyl-CoA, and n-butyryl-CoA). The initial velocity pattern using acetyl-CoA favors a ping-pong kinetic mechanism (Kia = -0.34 +/- 0.34 microM), but the pattern using n-propionyl-CoA supports a sequential one (Kia = 2.7 +/- 0.8 microM). Kinetic analyses using alternative substrates confirm the sequential mechanism and, moreover, clearly identify a random order of addition of antibiotic and coenzyme because V/K values of antibiotics varied 40-fold as the identity of the coenzyme was changed and V/K values of coenzyme varied 13-fold as the identity of the antibiotic was changed. One or both sets of values would have remained unchanged if the mechanism were either ordered sequential or ping-pong. Combining these results with structure-activity data which argue for a rapid rate of release of substrates and products relative to the rate of enzymatic turnover (Radika, K., and Northrop, D. B. (1984) Biochemistry 25, in press) establishes the kinetic mechanism as rapid equilibrium random sequential.

A polysaccharide composed of 3-O-methyl-D-mannose and D-mannose in a molar ratio of approximately 10:1 and containing 3 to 4 esterified acetyl residues has been isolated from Streptomyces griseus. This acetylated methylmannose polysaccharide (AMMP) is similar to the methylmannose polysaccharide (MMP) of Mycobacterium smegmatis (Gray, G. R., and Ballou, C. E. (1971) J. Biol. Chem. 246, 6835-6842) in its size and composition, the absence of acidic or basic groups, and the lack of a reducing end. It is different, however, in its content of esterified acetyl residues, and it is slightly different in its structure and in its gel filtration properties. The structure of AMMP has been established by proton magnetic resonance spectroscopy, and by combinations of methylation analysis and Smith degradation utilizing non-radioactively labeled polysaccharide and [3H]methyl-labeled polysaccharide obtained from cells grown in the presence of L-[methyl-3H]methionine. It is concluded that AMMP is a linear, nonreducing, neutral polysaccharide composed of a terminal D-mannose residue linked alpha(1 leads to 4) to a chain of 10 consecutive alpha(1 leads to 4)-linked 3-O-methyl-D-mannose residues. The reducing terminal 3-O-methyl-D-mannose residue exists, at least in part, as its alpha-methyl glycoside. The positions of attachment of the ester residues have not been established.

The 3-O-methyl-D-mannose-containing polysaccharide (MMP) from Mycobacterium smegmatis, first described by Gray and Ballou (Gray, G. R., and Ballou, C. E. (1971) J. Biol. Chem. 246, 6835-6842) is now shown to be a mixture of at least four isomers separable by gel filtration owing to differences in size and degree of methylation. The major component is 3-O-methylmannose but all contain small amounts of mannose. The molecular weights range from 2040 to 2490 and all are nonreducing. After Smith degradation, all yield a single large and one or more small fragments that give 3-O-methylmannose as the sole product of complete acid hydrolysis. The large Smith-degraded MMP components (SD-MMP) are similar to intact MMP and vary from 1830 to 2130 daltons, consistent with the loss of a single mannose; whereas the smaller fragments are the size of tri- to hexasaccharides and result from fragmentation of incompletely methylated chains. Controlled acid hydrolysis of [methyl-3H]MMP releases 6% of the methyl groups as [3H]methanol at a rate characteristic for the hydrolysis of methyl alpha-D-mannopyranoside. Proton magnetic resonance spectra of MMP and SD-MMP show a major methyl ether proton peak and a second small peak at higher field equivalent to about one methyl group per molecule. The results are consistent with the presence of an alpha-methyl aglycon at the reducing end of the chains. Methylation analysis of MMP isomers purified by high pressure liquid chromatography confirms that they are linear and unbranched. Methylation of [methyl-3H]MMP yields unlabeled tetra-O-methylmannose, showing that the chains are terminated by mannose. However, digestion of [methyl-3H]MMP with alpha-mannosidase releases mannose and exposes [methyl-3H]3-O-methylmannose. Smith degradation of [methyl-3H]MMP III yields a penta-to hexasaccharide product that can be resolved by high pressure liquid chromatography into two components. The distribution of radioactivity between these two fragments suggests that the chain was cleaved near the middle and that there must be an unmethylated mannose at that position. We conclude that the 3-O-methylmannose polysaccharides are linear unbranched chains of 11 to 14 sugar units, each terminated by a single mannose at the nonreducing end and by a methyl aglycon at the reducing end. Each isomer shows microheterogeneity, with 1 or 2 unmethylated mannose units near the middle of some but not all of the chains.

Lysosomal enzymes isolated from the slime mold Dictyostelium discoideum bind to the mannose 6-phosphate receptor which is present in many mammalian cells. While binding to the receptor suggests that the slime mold enzymes possess the same mannose 6-phosphate recognition marker as their mammalian counterparts, initial structural studies of the phosphorylated oligosaccharides have indicated that the phosphate is attached to high mannose-type units via an unusual phosphodiester linkage (Freeze, H.H., Yeh, R., Miller, A.L., and Kornfeld, S. (1983) J. Biol. Chem. 258, 14874-14879). To identify the components of the phosphodiester group we have isolated the phosphorylated high-mannose oligosaccharides from D. discoideum AX3 cells labeled with [2-3H]mannose or [6-3H]glucosamine and from the differentiation medium of unlabeled cells. The major phosphorylated species contain one or two phosphodiester groups and an average of 6 or 7 mannose residues. The phosphodiesters are relatively stable to both acid and base hydrolysis, but upon strong acid hydrolysis (conditions that completely hydrolyze the oligosaccharide) mannose 6-phosphate residues are liberated. Through a combination of techniques, including fast atom bombardment and direct chemical ionization mass spectrometry, it is shown that the mannose 6-phosphate residues of the intact oligosaccharide are diesterified to methyl groups. This indicates that slime mold possesses a different biosynthetic pathway for the formation of phosphorylated high mannose-type oligosaccharides than is utilized by higher organisms.