Analogs of ceramide that inhibit glucocerebroside synthetase in mouse brain

In a search for potent inhibitors of glucocerebroside biosynthesis, we synthesized aromatic analogs of the enzyme's substrate, ceramide, many of which have not previously been described in the literature. Mouse brain and spleen, rat brain, and human placenta and spleen were all found to be susceptible to inhibition by a variety of compounds, although to differing extents. The most potent inhibitor was 2-decanoylamino-3-morpholino-1-phenylpropanol. The dehydro version of this compound (2-decanoylamino-3-morpholinopropiophenone) was less effective but it produced inactivation of the enzyme, probably by covalent reaction with the enzyme's active site. Examination of the various effects seen leads us to suggest that the active region of the enzyme contains four recognitional sites: an anionic moiety that may bind the glucose in activated form, an oxygen-binding region oriented toward the third carbon atom of ceramide, a narrow region that binds the alkyl chain of the fatty acid moiety, and a less narrow region that binds the hydrocarbon chain of the sphingoid base moiety.     

Differentiation of β-glucocerebrosidase from β-glucosidase in human tissues using sodium taurocholate

Human tissues contain at least two enzymes capable of releasing glucose from 4-methylumbelliferyl-β-d-glucopyranoside, but only one of these enzymes can hydrolyze glucocerebroside and is deficient in individuals with Gaucher's disease. In the present report, we demonstrate that, in human liver, these two β-glucosidases differ in terms of their subcellular localization, chromatographic behavior on ion-exchange columns, substrate specificity, and sensitivity to inhibition or activation by sodium taurocholate and phospholipids. We also demonstrate that when the relatively nonspecific, artificial β-glucoside substrate, 4-methylumbelliferyl-β-d-glucopyranoside, is used under assay conditions optimal for glucocerebroside hydrolysis, it is effective in measuring relative glucocerebroside:β-glucosidase activity and can be used to evaluate an individual's status with respect to Gaucher's disease. These conditions of assay require a pH near neutrality (pH 5.5–6.5) and the presence of the detergent sodium taurocholate. The inclusion of sodium taurocholate in assays using 4-methylumbelliferyl-β-d-glucopyranoside as substrate permits the specific measurement of glucocerebroside:β-glucosidase activity because sodium taurocholate inhibits the nonspecific β-glucosidase not involved in Gaucher's disease and stimulates the relevant β-glucocerebrosidase activity.     

Inhibition of angiotensin-converting enzyme by angiotensin I analogue peptide inhibitors. A kinetic study

Angiotensin I analogues with a phosphonic acid group replacing the C-terminal carboxyl group were shown to be competitive inhibitors of angiotensin-converting enzyme. This new class of inhibitors was used to study the binding requirements of the angiotensin I-like ligands to the enzyme's active site. These studies indicate that angiotensin-converting enzyme recognizes at least five amino acid residues at the C-terminus of the peptide. The effect of pH on the binding of the most potent inhibitor peptide was compared to Captopril. The two inhibitors showed similar Ki-pH profiles despite their structural differences. Chloride enhanced the binding of the peptide inhibitor at both pH 9.0 and pH 6.5. At pH 9.0 the inhibitor peptide and the anion bind randomly to the enzyme, while at pH 6.5 the mechanism is ordered. In the latter case, the anion binds first to the enzyme.     

Cyclopentanoid analogs of phosphatidylcholine: susceptibility to phospholipase A2

Six isomers of dipalmitoylcyclopentanetriol phosphocholine (cyclopentano-lecithin) were tested as potential substrates for phospholipase A2. Since each of these analogs possesses a configuration that mimics a narrow range of conformations of a glycerophospholipid molecule, the analogs were used to assess the enzyme's conformational requirements. Studies showed that all of the analogs containing the phosphocholine at the C-1 (or C-3) position could be hydrolyzed, while only one of the three analogs that contains the polar head group at the C-2 position was susceptible. Kinetic studies, however, revealed that only the all-trans-(1,3/2-1P)-cyclopentano-lecithin gave initial rates of hydrolysis that were measurable by pH-stat. Acyl group specificity of the enzyme towards the all-trans isomer was determined with an analog was acyl groups were distinguishable. The synthesis of this mixed-acid-cyclopentano-PC is described herein. When this analog was enzymatically assayed, results unequivocally showed the enzyme to be specific for C-2 acyl hydrolysis. This specificity, and data showing that the all-trans analog is stereospecifically hydrolyzed, indicate that it is acted on in an analogous manner to dipalmitoylphosphatidylcholine. These studies indicate that although the configuration of the analog is not necessarily a prerequisite for hydrolysis, there does appear to be an optimal spatial orientation for enzymatic activity. The analogy between the susceptibilities of all-trans-(1,3/2-1P)-cyclopentano-lecithin and glycero-lecithin suggests that the conformation of the glycero-lecithin during phospholipase A2-mediated hydrolysis may be best simulated by the all-trans orientation of C-O bonds in the artificial substrate.     

Animal and cellular models of Gaucher's disease. I. Refractoriness of thio analogs of glucocerebroside to enzymatic hydrolysis

In order to develop a nonmetabolizable analog of glucocerebroside to investigate the distribution and accumulation of this lipid in model systems, thiohemiacetal derivatives were synthesized and their susceptibility to enzymatic hydrolysis by purified human placental glucocerebrosidase was examined. Sulfur analogs were found to be completely refractory to the activity of this enzyme, indicating their potential use in animal and isolated cell models and possibly for the preparation of affinity chromatography columns for the isolation of glucocerebrosidase.     

Thermotropic behavior of monoglucocerebroside--dipalmitoylphosphatidylcholine multilamellar liposomes

The thermotropic behavior of multilamellar liposomes prepared from mixtures of glucocerebroside and dipalmitoylphosphatidylcholine has been studied by high-sensitivity scanning calorimetry. It is shown that glucocerebroside has a marked effect on the gel--liquid crystalline transition of dipalmitoylphosphatidylcholine. The pretransition seen in pure samples of dipalmitoylphosphatidylcholine is undetectable at small mode fractions of glucocerebrosides (less than 10%). The main transition is shifted to higher temperatures and becomes broader and less cooperative in the presence of glucocerebroside. The enthalpy change of the main transition decreases with increasing the glucocerebroside content. However, this decrease is not linear with the glucocerebroside/phospholipid mole ratio. Glucocerebroside itself does not show a separate transition in the temperature range of these studies (10--75 degree C). The origin of these effects and their dependence on the glucocerebroside content suggest that the in-plane distribution of glucocerebroside molecules is affected by the physical state of the lipid bilayer and by the glucocerebroside/phospholipid mole ratio.     

Structural basis for substrate specificity of the human mitochondrial deoxyribonucleotidase

The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3'-monophosphate, thymidine 5'-monophosphate, deoxyuridine 5'-monophosphate, uridine 5'-monophosphate, deoxyguanosine 5'-monophosphate, uridine 2'-monophosphate, and the 5'-monophosphate of the nucleoside analog 3'-deoxy 2'3'-didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme's specificity for the deoxyribo form of nucleoside 5'-monophosphates is due to Ile-133, Phe-49, and Phe-102, which surround the 2' position of the sugar and cause an energetically unfavorable environment for the 2'-hydroxyl group of ribonucleoside 5'-monophosphates. The close binding of the 3'-hydroxyl group of nucleoside 5'-monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzyme.     

Niemann-Pick disease, Type C: evidence for the deficiency of an activating factor stimulating sphingomyelin and glucocerebroside degradation

1) Qualitative lipid analyses by thin-layer chromatography of 4 Niemann-Pick type C spleens confirmed sphingomyelin accumulation together with increase in the amount of glucocerebroside. 2) In the presence of crude sodium taurocholate as detergent, sphingomyelin degradation rates of normal and Niemann-Pick type C-cultured fibroblasts were fairly close under standard conditions at pH 5.0. In the absence of sodium taurocholate, sphingomyelinase activity was optimal at pH 4.0. Sphingomyelinase activities of fibroblasts from two patients with Niemann-Pick disease type C measured without detergent, were about 30% of that of controls. 3) Extracts from Gaucher spleen heated to 90 degrees C and devoid of sphingomyelinase activity stimulated at the optimal pH of 4.0 sphingomyelin degradation by cultured normal fibroblasts (2--4-fold, Niemann-Pick type C fibroblasts (5--9-fold), whereas similarly treated extracts from Niemann-Pick type C spleen showed no stimulation of sphingomyelin catabolism. Heated extracts from normal human spleen exhibited a smaller stimulation than that shown by Gaucher spleen. This stimulating effect could not be observed in fibroblasts from patients suffering from Niemann-Pick type B (sphingomyelinase defect). 4) Heat-treated extracts of Gaucher spleen were fractionated by ion exchange chromatography, isoelectric focusing and gel filtration. The active fractions obtained by these procedures stimulated sphingomyelin as well as glucocerebroside degradation and were absent from the corresponding Niemann-Pick type C preparations. Enriched activator preparations of Gaucher spleen stimulated sphingomyelinase activity of Niemann-Pick type C fibroblasts 25--38-fold and that of normal cells 3-fold. 5) The activating factor had an isoelectric point of 4.0 and an apparent molecular weight, as estimated by gel filtration, of 25000. Treatment with pronase E abolished its activity.     

The distribution of glucocerebroside in the liver of patients with Gaucher's disease.

Two Gaucher livers obtained at autopsy were examined for uniformity of glucocerebroside content. Six needle biopsies were obtained from one liver and twenty from the other, Samples were analyzed for glucocerebroside content by thin-layer and gas-liquid chromatography. One of the livers was partitioned into four sections with five biopsies taken at random from each section. This method supposes that if differences in glycolipid concentrations exist, then these differences will be exhibited among the liver sections. Our findings indicate that stored glucocerebroside is uniformly distributed throughout the liver since all the observed glycolipid levels were within ± 1.95 sample standard deviations of the sample mean.     

Identification of subsidiary catalytic groups at the active site of beta-ketoacyl-CoA thiolase by covalent modification of the protein

beta-Ketoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) is known to possess sulfhydryl groups of cysteines at the active site that are essential for its catalytic activity. Other groups at the active site that participate in the catalytic process were identified by using anhydride reagents which covalently modify the protein by specifically reacting with any amino groups potentially present at the active site. Since these reagents may also react with thiol groups, the enzyme's amino groups were modified after masking the cysteine thiols present by an alkylalkane thiosulfonate-type reagent, methyl methanethiol-sulfonate (MMTS), that selectively formed a disulfide bridge, thus generating an inactive thiolmethylated enzyme. When this procedure was followed, the enzyme could be undoubtedly modified at its amino by the anhydride reagent, leading to a doubly modified protein. The thiomethyl group could then be removed by reduction with dithiothreitol, yielding an enzyme modified solely on the amino residues. The amino group could be unblocked in turn by exposure to acidic pH. The different anhydrides inactivated thiolase, but only acetoacetyl coenzyme A (AcAcCoA) provided any protection against inactivation. When thiolmethylcitraconyl thiolase was reduced with dithiothreitol the enzyme remained inactive, but when the doubly modified enzyme was exposed to pH 5 then the reduction led to formation of an active enzyme. These results are interpreted as demonstrating a role for an amino group at the enzyme active site. A catalytic mechanism is proposed for the enzyme which involves the amino group.     

Dichlorophenolindophenol-linked formate dehydrogenase of the methanol-utilizing Mycobacterium gastri MB19

Abstract 2,6-Dichlorophenolindophenol (DCIP)-linked formate dehydrogenase activity has been demonstrated for the first time in the cell-free extract of a methylotrophic mycobacterium, Mycobacterium gastri MB19. The enzyme was produced when the strain was cultivated with methanol, glucose or mannitol as a carbon source, whereas no enzyme production occurred with other multi-carbon compounds. The enzyme was located in the particulate fraction. Although the enzyme was unstable on preservation at 4° C in potassium phosphate buffer (pH 7.0), it was stabilized under acidic conditions (pH 5.0). Glycerol and EDTA were also effective for the enzyme's stability. The optimum pH and temperature for the enzyme's activity were 7.0° and 55° C, respectively.     

Purification and properties of citrate lyase from Streptococcus diacetilactis.

Citrate lyase from Streptococcus diacetilactis has been purified to yield a protein that was homogeneous as judged by sedimentation velocity and sedimentation equilibrium experiments. The enzyme's sedimentation coefficient is 16.8 S and its molecular weight is around 585,000. It contains three nonidentical subunits of about 53,000, 34,000, and 10,000 daltons. The enzyme in its active form contains an acetyl group which turns over during the citrate cleavage reaction. Removal of the acetyl group inactivates the enzyme. The deacetyl enzyme can be partially reactivated by acetylation with acetic anhydride. The enzyme undergoes slow "reaction-inactivation." The rate of inactivation is first order and the rate constant of inactivation is much lower than that for a similar inactivation process of the citrate lyase from Klebsiella aerogenes. Like the latter enzyme it contains stoichiometric amounts of phosphopantothenate. The enzyme is inactivated at pH greater than 8.1 and the presence of citrate provides protection against this inactivation. Sedimentation studies of the enzyme at pH 8.7 indicate that the enzyme is dissociated, which may account for the inactivation. The enzyme is immunologically different from citrate lyases of K. aerogenes and Escherichia coli.     

Expression and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the rhizobacterium Pseudomonas putida UW4: a key enzyme in bacterial plant growth promotion

The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) converts ACC, the precursor of the plant hormone ethylene, to alpha-ketobutyrate and ammonium. This enzyme has been identified in soil bacteria and has been proposed to play a key role in microbe-plant association. A soluble recombinant ACCD from Pseudomonas putida UW4 of molecular weight 41 kDa has been cloned, expressed, and purified. It showed selectivity and high activity towards the substrate ACC: K(M)=3.4+/-0.2 mM and k(cat)=146+/-5 min(-1) at pH 8.0 and 22 degrees C. The enzyme displayed optimal activity at pH 8.0 with a sharp decline to essentially no activity below pH 6.5 and a slightly less severe tapering in activity at higher pH resulting in loss of activity at pH>10. The major component of the enzyme's secondary structure was determined to be alpha-helical by circular dichroism (CD). P. putida UW4 ACCD unfolded at 60 degrees C as determined by its CD temperature profile as well as by differential scanning microcalorimetry (DSC). Enzyme activity was knocked out in the point mutant Gly44Asp. Modeling this mutation into the known yeast ACCD structure shed light on the role this highly conserved residue plays in allowing substrate accessibility to the active site. This enzyme's biochemical and biophysical properties will serve as an important reference point to which newly isolated ACC deaminases from other organisms can be compared.     

The Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with laevulinic acid.

The X-ray structure of the complex formed between yeast 5-aminolaevulinic acid dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15 A resolution. The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263. It is known that this lysine forms a Schiff base link with substrate bound at the enzyme's so-called P-site. The carboxyl group of laevulinic acid makes hydrogen bonds with the side-chain-OH groups of Tyr329 and Ser290, as well as with the main-chain >NH group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site. Our analysis strongly suggests that the same interactions will be made by P-side substrate and also indicates that the substrate that binds at the enzyme's A-site will interact with the enzyme's zinc ion bound by three cysteines (133, 135, and 143). Inhibitor binding caused a substantial ordering of the active site flap (residues 217-235), which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis.     

Cathepsin B stability,but not activity,is affected in cysteine:cystine redox buffers

In order to test the hypothesis that the lysosomal cysteine protease cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine- and/or cystine-containing buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0-7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. However, at pH 7.0, the stability of cathepsin B decreased with increasing reduction potential and ambient cystine concentration. This suggests that the stability of the enzyme at neutral pH is dependent on redox potential, and on the presence of oxidising agents.     

Interactions of monodispersed and micellar substrates with a phospholipase A2 from Trimeresurus flavoviridis

Bindings of the phospholipase A2 from Trimeresurus flavoviridis to the monodispersed and micellar n-alkylphosphorylcholines (n-CnPC) were studied at 25 degrees C and ionic strength 0.2 by the aromatic CD and tryptophyl fluorescence methods, respectively. The bindings to micelles of the substrate analog were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 9-13. The binding constant to the micelle was about 40 times greater than it was to the monodispersed substrate. The binding constant to the micellar substrate analog increased on the binding of Ca2+ to the enzyme and decreased on modification of the N-terminal alpha-NH2 group, whereas the binding to the monodispersed substrate analog was independent of pH, of the Ca2+ binding, and of the chemical modification of the alpha-NH2 group. The kinetics of the hydrolyses of monodispersed and micellar dihexanoylphosphatidylcholines (diC6PC) were studied at 25 degrees C and ionic strength 0.2 by the pH-stat method in the presence of saturating amounts of Ca2+. The catalytic center activity, kappa cat, as well as the binding constant, 1/Km, for the micellar substrate, were found to be much greater than those for the monodispersed substrate. The binding constant, 1/Km, of the monodispersed substrate was independent of pH; this was in good agreement with that of the substrate analog described above. The pH-dependence curve of kappa cat for the monodispersed substrate exhibited two transitions, one below pH 6.5 and the other above pH 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of rates of hydrolysis of N-oleoyl and N-stearoyl glucocerebroside in patients with Gaucher's disease

The deficiency of oleic acid as one of the fatty acids in glucocerebrosides that accumulate (31--77 mg/g dry weight) in the spleen in patients with Gaucher's disease was confirmed in 9 cases. In an effort to account for the 10-fold difference between the oleoyl glycocerebroside content of glucocerebrosides in spleen from controls and patients with Gaucher's disease, we compared the ability of extracts of spleen and fibroblasts from individuals with various forms of Gaucher's disease and controls to hydrolyze [14C]stearoyl and [3H]oleoyl glucocerebroside. The residual glucosylceramidase activity in patients with Gaucher's disease hydrolyzes the glucose moiety of oleoyl glucocerebroside at approximately the same rate as that of stearoyl glucocerebroside. Similarly, the more active glucosylceramidase of control tissue acts upon both oleoyl and stearoyl glucocerebrosides with equal efficiency. These observations indicate that a mutation affecting the substrate specificity of glucosylceramidase cannot account for the lack of oleic acid-containing glucocerebrosides in patients with Gaucher's disease. Thus, the hypothesis that the difference in fatty acid composition found in glucocerebroside is obtained as a result of a mutation affecting the specificity of the residual glucosylceramidase must be rejected.     

Identification and characterization of the acid phosphatase HppA in Helicobacter pylori

An acid phosphatase (HppA) activated by NH4Cl was purified 192- and 34-fold from the periplasmic and membrane fractions of Helicobacter pylori, respectively. SDS-polyacrylamide gel electrophoresis revealed that HppA from the latter appears to be several kilodaltons larger in molecular mass than from the former by about 24 kDa. Under acidic conditions (pH< or =4.5), the enzyme activity was entirely dependent on the presence of certain mono- and/or divalent metal cations (e.g., K+, NH4 +, and/or Ni2+). In particular, Ni2+ appeared to lower the enzyme's Km for the substrates, without changing Vmax. The purified enzyme showed differential specificity against nucleotide substrates with pH; for example, the enzyme hydrolyzed adenosine nucleotides more rapidly at pH 5.5 than at pH 6.0, and vice versa for CTP or TTP. Analyses of the enzyme's N-terminal sequence and of an HppA- H. pylori mutant revealed that the purified enzyme is identical to rHppA, a cloned H. pylori class C acid phosphatase, and shown to be the sole bacterial 5'-nucleotidase uniquely activated by NH4Cl. In contrast to wild type, HppA- H. pylori cells grew more slowly. Strikingly, they imported Mg2+ at a markedly lowered rate, but assimilated urea rapidly, with a subsequent increase in extracellular pH. Moreover, mutant cells were much more sensitive to extracellular potassium ions, as well as to metronidazole, omeprazole, or thiophenol, with considerably lowered MIC values, than wild-type cells. From these data, we suggest that the role of the acid phosphatase HppA in H. pylori may extend beyond 5'-nucleotidase function to include cation-flux as well as pH regulation on the cell envelope.     

alpha-Bromo-4-amino-3-nitroacetophenone, a new reagent for protein modification. Modification of the methionine-290 residue of porcine pepsin.

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.     

pH-dependent inhibitory effects of tris and lithium ion on intestinal brush-border sucrase

Tris and two of its hydroxylated amine analogs were examined in a metal-free, universal n-butylamine buffer, for their interaction with intestinal brush border sucrase. Our recent three-proton-families model (Vasseur, van Melle, Frangne and Alvarado (1988) Biochem. J., 251, 667-675) has provided the sucrase pK values necessary to interpret the present work. At pH 5.2, 2-amino-2-methyl-l-propanol (PM) causes activation whereas Tris has a concentration-dependent biphasic effect, first causing activation, then fully competitive inhibition. The amine species causing activation is the protonated, cationic form. The difference between the two amines is related to the fact that Tris has a much lower pKa value than PM (respectively, 8.2 and 9.8). Even at pH 5.2, Tris (but not PM) exists as a significant proportion of the free base which, by inhibiting the enzyme fully competitively, overshadows the activating effect of the cationic, protonated amine. Above pH 6.8, both Tris and PM act as fully competitive inhibitors. These inhibitions increase monotonically between pH 6.5 and 8.0 but, above pH 8, inhibition by 2.5 mM Tris tends to diminish whereas inhibition by 40 mM PM increases abruptly to be essentially complete at pH 9.3 and above. As pH increases from 7.6 to 9.0, the apparent affinity of the free amine bases decreases whereas that of the cationic, protonated amines, increases. In this way, the protonated amines replace their corresponding free bases as the most potent inhibitors at high pH. The pH-dependent inhibition by 300 mM Li+ is essentially complete at pH 8, independent of the presence or absence of either 2.5 mM Tris or 40 mM PM. Even at pH 7.6, an excess (300 mM) of Li+ causes significant increases in the apparent Ki value of each Tris, PD (2-amino-2-methyl-1-3-propanediol) and PM, suggesting the possibility of a relation between the effects of Li+ and those of the hydroxylated amines which in fact are mutually exclusive inhibitors. The inhibitory results are interpreted in terms of a mechanistic model in which the free bases bind at two distinct sites in the enzyme's active center. Binding at the glucosyl sub-site occurs through the amine's free hydroxyl groups. This positioning facilitates the interaction between the lone electron pair of the deprotonated amino group with a proton donor in the enzyme's active center, characterized by a pK0 around 8.1. When this same group deprotonates, then the protonated amines acting as proton donors replace the free bases as the species giving fully competitive inhibition of sucrase.