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Structure-function relationship of xylanase: fluorimetric analysis of the tryptophan environment.

ABSTRACT: Involvement of one out of three tryptophan residues in the active site of the low-molecular-mass xylanase from Chainia has been demonstrated previously [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172-177]. The work described here aims at: (i) deducing the structure-function relationship for the tryptophan residue involved at the active site (a) by correlating the effect of N-bromosuccinimide (NBS) on the fluorescence and activity, and (b) by assessing the ability of xylan to protect against decrease in fluorescence versus activity of NBS-treated enzyme; and (ii) probing into the environment of the tryptophan residues by studying the quenching of their fluorescence by various solute quenchers in the presence and absence of guanidine hydrochloride (Gdn.HCl). Complete inactivation of the NBS-treated enzyme occurs well before the loss of fluorescence. Full protection by xylan (0.5%) of the inactivation of enzyme by NBS compared with 30% protection for the decrease in fluorescence confirms the participation of a single tryptophan at the substrate-binding site of the xylanase. The xylanase exhibited a rather low fluorescence emission maximum at 310 nm. There was no shift in the emission maximum on treatment of the enzyme with Gdn.HCl (6.5 M), indicating the rigidity of the microenvironment around tryptophan residues. The quenching studies with acrylamide suggested the occurrence of both collisional as well as static quenching processes. The enzyme retained full activity as well as the characteristic emission maximum at 310 nm in the presence of acrylamide (100 mM), indicating that quenching of fluorescence by acrylamide is a physical process. Acrylamide was more efficient as a quencher than CsCl or KBr. Treatment of the enzyme with Gdn.HCl resulted in an increase in accessibility of the quenchers to the fluorophore as suggested by an increase in the Stern-Volmer quenching constants (K(SV)) of the solute quenchers. The analysis of K(SV) and V values of KBr and CsCl suggests that the overall tryptophan microenvironment in the xylanase from Chainia is slightly electronegative.

SUBMITTER: Bandivadekar KR

PROVIDER: S-EPMC1217236 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other

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Project description:The monovalent cation (MVC) site of the tryptophan synthase from Salmonella typhimurium plays essential roles in catalysis and in the regulation of substrate channeling. In vitro, MVCs affect the equilibrium distribution of intermediates formed in the reaction of l-Ser with the alpha(2)beta(2) complex; the MVC-free, Cs(+)-bound, and NH(4)(+)-bound enzymes stabilize the alpha-aminoacrylate species, E(A-A), while Na(+) binding stabilizes the l-Ser external aldimine species, E(Aex(1)). Two probes of beta-site reactivity and conformation were used herein, the reactive indole analogue, indoline, and the l-Trp analogue, l-His. MVC-bound E(A-A) reacts rapidly with indoline to give the indoline quinonoid species, E(Q)(indoline), which slowly converts to dihydroiso-l-tryptophan. MVC-free E(A-A) gives very little E(Q)(indoline), and turnover is strongly impaired; the fraction of E(Q)(indoline) formed is <3.5% of that given by the Na(+)-bound form. The reaction of l-Ser with the MVC-free internal aldimine species, E(Ain), initially gives small amounts of an active E(A-A) which converts to an inactive species on a slower, conformational, time scale. This inactivation is abolished by the binding of MVCs. The inactive E(A-A) appears to have a closed beta-subunit conformation with an altered substrate binding site that is different from the known conformations of tryptophan synthase. Reaction of l-His with E(Ain) gives an equilibrating mixture of external aldimine and quinonoid species, E(Aex)(his) and E(Q)(his). The MVC-free and Na(+) forms of the enzyme gave trace amounts of E(Q)(his) ( approximately 1% of the beta-sites). The Cs(+) and NH(4)(+) forms gave approximately 17 and approximately 14%, respectively. The reactivity of MVC-free E(Ain) was restored by the binding of an alpha-site ligand. These studies show MVCs and alpha-site ligands act synergistically to modulate the switching of the beta-subunit from the open to the closed conformation, and this switching is crucial to the regulation of beta-site catalytic activity. Comparison of the structures of Na(+) and Cs(+) forms of the enzyme shows Cs(+) favors complexes with open indole binding sites poised for the conformational transition to the closed state, whereas the Na(+) form does not. The beta-subunits of Cs(+) complexes exhibit preformed indole subsites; the indole subsites of the open Na(+) complexes are collapsed, distorted, and too small to accommodate indole.

Project description:BACKGROUND:Sequence similarity to characterized proteins provides testable functional hypotheses for less than 50% of the proteins identified by genome sequencing projects. With structural genomics it is believed that structural similarities may give functional hypotheses for many of the remaining proteins. METHODOLOGY/PRINCIPAL FINDINGS:We provide a systematic analysis of the structure-function relationship in proteins using the novel concept of local descriptors of protein structure. A local descriptor is a small substructure of a protein which includes both short- and long-range interactions. We employ a library of commonly reoccurring local descriptors general enough to assemble most existing protein structures. We then model the relationship between these local shapes and Gene Ontology using rule-based learning. Our IF-THEN rule model offers legible, high resolution descriptions that combine local substructures and is able to discriminate functions even for functionally versatile folds such as the frequently occurring TIM barrel and Rossmann fold. By evaluating the predictive performance of the model, we provide a comprehensive quantification of the structure-function relationship based only on local structure similarity. Our findings are, among others, that conserved structure is a stronger prerequisite for enzymatic activity than for binding specificity, and that structure-based predictions complement sequence-based predictions. The model is capable of generating correct hypotheses, as confirmed by a literature study, even when no significant sequence similarity to characterized proteins exists. CONCLUSIONS/SIGNIFICANCE:Our approach offers a new and complete description and quantification of the structure-function relationship in proteins. By demonstrating how our predictions offer higher sensitivity than using global structure, and complement the use of sequence, we show that the presented ideas could advance the development of meta-servers in function prediction.

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Structure-function relationship of xylanase: fluorimetric analysis of the tryptophan environment.

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