Structure, dynamics, and stability of beta-cyclodextrin inclusion complexes of aspartame and neotame.

Studies of the high-intensity sweetener aspartame show that its stability is significantly enhanced in the presence of beta-cyclodextrin (beta-CyD). At a 5:1 beta-CyD/aspartame molar ratio, the stability of aspartame is 42% greater in 4 mM phosphate buffer (pH 3.1) compared to solutions prepared without beta-CyD. Solution-state (1)H NMR experiments demonstrate the formation of 1:1 beta-CyD/aspartame complexes, stabilized by the interaction of the phenyl-ring protons of aspartame with the H3 and H5 protons of beta-CyD. Inclusion complex formation clearly accounts for the observed stability enhancement of aspartame in solution. The formation of inclusion complexes in solution is also demonstrated for beta-CyD and neotame, a structural derivative of aspartame containing an N-substituted 3,3-dimethylbutyl group. These complexes are stabilized by the interaction of beta-CyD with both phenyl-ring and dimethylbutyl protons. Solid-state NMR experiments provide additional characterization, clearly demonstrating the formation of inclusion complexes in lyophilized solids prepared from solutions of beta-CyD and either aspartame or neotame.

Interconversion of the ligand arrays in the CD and EF sites of oncomodulin. Influence on Ca2+-binding affinity.

The parvalbumin metal ion-binding sites differ at the +z and -x residues: Whereas the CD site employs serine and glutamate (or aspartate), respectively, the EF site employs aspartate and glycine. Although frequently indistinguishable in Ca2+- and Mg2+-binding assays, the CD and EF sites nonetheless exhibit markedly different preferences for members of the lanthanide series [Williams et al. (1984) J. Am. Chem. Soc. 106, 5698-5702], underscoring an intrinsic nonequivalence. This nonequivalence reaches its pinnacle in the mammalian beta-parvalbumin (oncomodulin). Whereas the oncomodulin EF site exhibits the expected Ca2+/Mg2+ signature, the Ca2+ affinity of the CD site is severely attenuated. To obtain insight into the structural factors responsible for this reduction in binding affinity, oncomodulin variants were examined in which the CD and EF site ligand arrays had been exchanged. Our data suggest that binding affinity may be dictated either by ligand identity or by the binding site environment. For example, the Ca2+ affinity of the quasi-EF site resulting from the combined S55D and D59G mutations is substantially lower than that of the authentic EF site. This finding implies that other local environmental variables (e.g., binding loop flexibility, electrostatic potentials) within the CD binding site supersede the influence of ligand identity. However, the CD site ligand array does not acquire a high-affinity signature when imported into the EF site, as in the D94S/G98D variant. Instead, it retains its Ca2+-specific signature, implying that this constellation of ligands is less sensitive to placement within the protein molecule. The D59G and D94S single mutations substantially lower binding affinity, consistent with removal of a liganding carboxylate. By contrast, the S55D and G98D mutations substantially increase binding affinity, a finding at odds with corresponding data collected on model peptide systems. Significantly, the Ca2+ affinity of the oncomodulin CD site is increased by mutations that weaken binding at the EF site, indicating a negatively cooperative interaction between the two sites.

1H-n.m.r. studies of squash seed trypsin inhibitor.

1H-n.m.r. studies at 500 MHz have been performed on a trypsin inhibitor (CMTI-III) found in squash seed (Cucurbita maxima). The sequential resonance assignments have been made using two-dimensional techniques. The chemical shifts for the assigned protons are reported at 30 degrees, pH 2.8 and form a basis for the determination of the solution structure of CMTI-III. Analysis of the NOE data, NH-alpha CH vicinal coupling constants and pattern of slowly exchanging amide protons indicates that the predominant feature of the solution conformation is a triple stranded beta sheet consisting of residues 8-10, 21-23, and 26-29. Residues 12-15 appear to form a beta turn.

Mechanism of the EPSP synthase catalyzed reaction: evidence for the lack of a covalent carboxyvinyl intermediate in catalysis.

In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of [14C] from [14C]-S3P and [14C]-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange.

Chemistry of the inactivation of cytosolic aspartate aminotransferase by serine O-sulfate.

The reaction of serine O-sulfate with cytosolic aspartate aminotransferase [John, R.A., & Fasella, P. (1969) Biochemistry 8, 4477] has been reinvestigated. As in the corresponding reaction with beta-chloroalanine [Morino, Y., Osman, A.M., & Okamoto, M. (1974) J. Biol. Chem. 249, 6684], the enzyme is inactivated over a 10-min period, and the absorption maximum at pH 5.4 shifts from 430 to 336 nm. Upon prolonged standing the peak shifts again over a period of 20 h to 455 nm, a behavior entirely similar to that reported by Morino et al. for beta-chloroalanine in the presence of 3 M formate. When the pH of either the 10-min product (1a) or the 20-h product (1b) is raised to 11 or above, a yellow, diffusible compound (2) is released from the protein. This compound as well as its dephosphorylation and reduction products has been isolated and studied by NMR spectroscopy. Compound 2 is identical with a compound formed from serine sulfate and glutamate decarboxylase by a similar reaction sequence [Likos, J.J., Ueno, H., Feldhaus, R.W., & Metzler, D.E. (1982) Biochemistry (preceding paper in this issue)] and is the product of an aldol condensation of pyruvate with pyridoxal phosphate. When the 20-h product 1b is reduced with sodium borohydride and then heated in a boiling water bath, a material identical with the reduction product of 2 is released. We propose that the 20-h product 1b consists of 2 bound to the enzyme. Pathways for the formation of the various compounds are proposed. These findings require a reevaluation of the mechanisms of action of many enzyme-activated inhibitors of pyridoxal phosphate dependent enzymes.

Affinity labeling of the active site of yeast pyruvate kinase by 5'-p-fluorosulfonylbenzoyl adenosine.

Yeast pyruvate kinase is irreversibly inactivated by 1.1 mM 5'-p-fluorosulfonylbenzoyl adenosine at pH 8.6 with an initial rate constant of 0.019 min-1. A plot of kinact versus the 5'-p-fluorosulfonylbenzoyl adenosine concentration yields a hyperbolic curve indicative of binding of the analog prior to reaction. Marked protection is afforded by phosphoenolpyruvate + fructose 1,6-diphosphate + Mg2+ or MgATP suggesting that reaction occurs within the active site. When assayed at less than saturating phosphoenolpyruvate concentrations, the inactivation caused by the reagent in the absence of added ligands appears slower, and reaction in the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+ produces an activation of the enzyme, the extent of which is dependent on the assay concentration of phosphoenolpyruvate. The rate constant for activation was observed to be 0.113 min-1. The activated enzyme exhibits both a lowered K0.5 and Hill coefficient compared to native pyruvate kinase. Subsequent addition of 5'-p-fluorosulfonylbenzoyl adenosine to activated pyruvate kinase in the absence of added ligands leads to inactivation with the rate constant independent of the assay concentration of phosphoenolpyruvate. Covalent reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyl adenosine thus occurs at two distinct sites. In the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+, incorporation of tritiated 5'-p-fluorosulfonylbenzoyl adenosine is linearly proportional to the extent of activation of the enzyme, with 4 mol of reagent bound/mol of tetrameric pyruvate kinase for maximally activated enzyme. In the absence of added ligands, approximately 4.5 mol of reagent are incorporated/mol of enzyme at 15 min of reaction, while 80% of the original activity remains. Subsequent incorporation is proportional to the extent of inactivation with 8 mol bound at 100% in activaton. In the presence of phosphoenolpyruvate, fructose 1,6-diphospate, and Mg2+, 3 tyrosines and 1 lysine residue, and in the absence of ligands, 6 tyrosines and 2 lysine residues are modified, suggesting that both amino acids are within the two nucleotide sites.

Hydrolysis of casein: a differential aid for the indentification of Serratia marcescens.

Through the hydrolysis of casein, a cultural characteristic has been established in the preliminary identification of Serratia marcescens. Compared against 438 strains of the families, Pseudomonadaceae and Enterobacteriaceae, this organism has a reproducible capability to decompose casein, while most other Gram-negative bacilli failed to produce any hydrolysis, or, at the most, hazy zones only. Routine use has shown this to be a valuable differential aid in the identification of Serratia marcescens.