Acid-base Residues Research Articles

Molecular characterization of a novel beta-glucuronidase from Scutellaria baicalensis georgi.

We cloned a gene encoding Scutellaria beta-glucuronidase (sGUS) that is involved in the initiation of H(2)O(2) metabolism in skullcap (Scutellaria baicalensis). This gene consists of a 1581-nucleotide open reading frame, the deduced amino acid sequence of which contains an ATP/GTP binding site and a leucine zipper motif. sGUS has apparent similarity to the heparan sulfate-metabolizing beta-glucuronidase heparanase but no homology to family 2 beta-glucuronidases. In addition, neither the family 2 glycosylhydrolase signature nor family 2 acid-base catalyst was found in this enzyme. These results suggested that sGUS does not belong to the family 2 beta-glucuronidases. We modified several residues predicted to act as the acid-base or nucleophilic residue of sGUS by site-directed mutagenesis. Mutations at Glu(212) or Glu(329) resulted in much lower k(cat)/K(m) values in the mutants as compared with the wild-type enzyme, indicating that these are the acid-base and nucleophilic residues of the active site, respectively. Moreover, similar site-directed mutagenesis confirmed that Tyr(281) is also involved in the beta-glucuronidase activity. The amino acid sequences of small regions containing these active site residues were conserved in heparanases. As sGUS has various structural characteristics in common with heparanase, we concluded that sGUS and heparanase belong to the same new family.

Active Site Residues of Human β-Glucuronidase: EVIDENCE FOR GLU540 AS THE NUCLEOPHILE AND GLU451 AS THE ACID-BASE RESIDUE

Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose that residues Glu(451), Glu(540), and Tyr(504) in hGUSB are involved in catalysis, Glu(451) being the acid-base residue and Glu(540) the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu(451), Glu(540), and Tyr(504) hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. The k(cat)/K(m) values (mM(-1).s(-1)) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu(451) as the acid-base catalyst. We conclude that, like their homologues in E. coli beta-galactosidase, Glu(540) is the nucleophilic residue, Glu(451) the acid-base catalyst, and Tyr(504) is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB.

Probing the Mechanism of Bacillus 1,3-1,4-β-d-Glucan 4-Glucanohydrolases by Chemical Rescue of Inactive Mutants at Catalytically Essential Residues

The role of the key catalytic residues Glu134 and Glu138 in the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis is probed by a chemical rescue methodology based on enzyme activation of inactive mutants by the action of added nucleophiles. While Glu134 was proposed as the catalytic nucleophile on the basis of affinity labeling experiments, no functional proof supported the assignment of Glu138 as the general acid-base catalyst. Alanine replacements are prepared by site-directed mutagenesis to produce the inactive E138A and E134A mutants. Addition of azide reactivates the mutants in a concentration-dependent manner using an activated 2, 4-dinitrophenyl glycoside substrate. The chemical rescue operates by a different mechanism depending on the mutant as deduced from 1H NMR monitoring and kinetic analysis of enzyme reactivation. E138A yields the beta-glycosyl azide product arising from nucleophilic attack of azide on the glycosyl-enzyme intermediate, thus proving that Glu138 is the general acid-base residue. Azide activates the deglycosylation step (increasing kcat), but it also has a large effect on a previous step (as seen by the large decrease in KM, the increase in kcat/KM, and the pH dependence of activation), probably increasing the rate of glycosylation through Bronsted acid catalysis by enzyme-bound HN3. By contrast, azide reactivates the E134A mutant through a single inverting displacement to give the alpha-glycosyl azide product, consistent with Glu134 being the catalytic nucleophile. Formate as an exogenous nucleophile has no effect on the E138A mutant, whereas it is a better activator of E134A than azide. Although the reaction yields the normal hydrolysis product, a transient compound was detected by 1H NMR, tentatively assigned to the alpha-glycosyl formate adduct. This is the first case where a nonmodified sugar gives a long-lived covalent intermediate that mimics the proposed glycosyl-enzyme intermediate of retaining glycosidases.

A program to calculate the isoelectric point of macromolecules

A program containing 260 sentences, written in BASIC and adapted to be run in personal computers, has been developed to calculate isoelectric point values of macromolecules (proteins, nucleic acids, polysaccharides, etc.). This implies the calculation of the coefficients and roots of a particular kind of polynomial. With common personal computers and using a compiled version of the program, the time required to calculate p I values of macromolecules containing for example, 9, 12, and 18 acid-base residues with different p K values was around 10 s, 2 min, and 2 h respectively.

Theoretical isoelectric points and electric charges of mutated human hemoglobin subunits

Isoelectric point values have been determined from a theoretical standpoint for α, β, γ and δ human abnormal subunits bearing elimination or addition of 1–2 different aminoacids with acid-base residues. The charge distribution, in relation to pH values, of the four types of subunits are also reported. These values may be of interest both to predict the electrophoretic behaviour and to characterize human abnormal hemoglobins.

An algorithm for the computer calculation of the coefficients of a polynomial that allows determination of isoelectric points of proteins and other macromolecules

The isoelectric point (p I) of a macromolecule containing any number of acid-base residues can be expressed as a polynomial whose coefficients are related to both the number of acid-base residues present in the molecule and their K values. Polynomials of degree higher than 5 are too complicated to be solved manually. An algorithm is here presented which allows, with few sentences written in BASIC, the calculation of the polynomial coefficients. With this knowledge and using standard polynomial solving programs, the isoelectric point of the macromolecule can be calculated.

Isoelectric points of proteins: Theoretical determination

Three methods for calculating the isoelectric points (p I) of proteins, provided that their amino acid compositions are known, are described. The comprehensive and abridged procedures involve solutions of polynomial equations of different degrees depending on whether p K values of the specific acid-base residues or the means of some of those values, respectively, are adopted. In the simplified procedure, approximate p I values of proteins can be determined easily with the help of calculated values, included in this paper, related to the amino acid composition of proteins.