Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding

T4 endonuclease V incises DNA at the sites of pyrimidine dimers through a two-step mechanism. These breakage reactions are preceded by the scanning of nontarget DNA and binding to pyrimidine dimers. In analogy to the synthetic tripeptides Lys-Trp-Lys and Lys-Tyr-Lys, which have been shown to be capable of producing single-strand scissions in DNA containing apurinic sites, endonuclease V has the amino acid sequence Trp-Tyr-Lys-Tyr-Tyr (128-132). Site-directed mutagenesis of the endonuclease V gene, denV, was performed at the Tyr-129 and at the Tyr-129 and Tyr-131 positions in order to convert the Tyr residues to nonaromatic amino acids to test their role in dimer-specific binding. The UV survival of repair-deficient (uvrA recA) Escherichia coli cells harboring the denV N-129 construction was dramatically reduced relative to wild-type denV+ cells. The survival of denV N-129,131 cells was indistinguishable from that of the parental strain lacking the denV gene. The mutant endonuclease V proteins were then characterized with regard to (1) dimer-specific nicking activity, (2) apurinic nicking activity, and (3) binding affinity to UV-irradiated DNA. Dimer-specific nicking activity and dimer-specific binding for both denV N-129 and N-129,131 were abolished, while apurinic-specific nicking was substantially retained in denV N-129,131 but was abolished in denV N-129. These results indicate that Tyr-129 and Tyr-131 positions of endonuclease V are at least important in pyrimidine dimer-specific binding and possibly nicking activity.     

Mutations in endonuclease V that affect both protein-protein association and target site location

A general mechanism by which proteins locate their target sites within large domains of DNA is a one-dimensional facilitated diffusion process in which the protein scans DNA in a nonspecifically bound state. An electrostatic contribution to this type of mechanism has been previously established. This study was designed to question whether other characteristics of a protein's structure might contribute to the scanning mechanism of target site location. In this regard, T4 endonuclease V was shown to establish an ionic strength dependent monomer-dimer equilibrium in solution. A protein dimer interaction site was postulated to exist along a putative alpha-helix containing amino acid residues 54-62. The conservative substitutions of Phe-60----Leu-60 and Phe-59, Phe-60----Leu-59, Leu-60 resulted in mutant enzymes which remained in the monomeric state independent of the ionic strength of the solution. The target site location mechanism of these mutants has also been altered. Under conditions where wild-type endonuclease V processively scans nontarget DNA, the target location mechanism of the monomeric mutant proteins was shifted toward a less processive search. This decrease in the processivity of the mutants was especially surprising because the nontarget DNA binding affinity was found to be significantly increased. Thus, an additional component of the endonuclease V DNA scanning mechanism appears to be the formation of a stable endonuclease V dimer complex.     

Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.     

Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase

Aspartyl aminopeptidase (DAP), a widely distributed and abundant cytosolic enzyme, removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. DAP is a member of the M18 family of the MH clan of cocatalytic metallopeptidases. The human and mouse enzymes have been cloned. We have identified 8 highly homologous eukaryotic sequences that are probable aspartyl aminopeptidases. Eight histidine residues of human DAP were sequentially mutated to phenylalanine. Mutation of His94, His170, and His440 abolished enzymatic activity. His94 and His440 are postulated to be involved in binding cocatalytic zinc atoms by homology with other members of the MH clan. Mutation of His352 dramatically reduced enzyme activity. Gel-filtration analysis of the His352 mutant revealed destabilization of the quaternary structure and dissociation of the native 440-kDa enzyme. Mutation of His33 and of histidines residing in a cluster at residues 349, 359, and 363 all decreased k(cat). These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.     

Catalytic mechanism of endonuclease v: a catalytic and regulatory two-metal model

The enzyme endonuclease V initiates repair of deaminated DNA bases by making an endonucleolytic incision on the 3' side one nucleotide from a base lesion. In this study, we have used site-directed mutagenesis to characterize the role of the highly conserved residues D43, E89, D110, and H214 in Thermotoga maritima endonuclease V catalysis. DNA cleavage and Mn(2+)-rescue analysis suggest that amino acid substitutions at D43 impede the enzymatic activity severely while mutations at E89 and D110 may be tolerated. Mutations at H214 yield enzyme that maintains significant DNA cleavage activity. The H214D mutant exhibits little change in substrate specificity or DNA cleavage kinetics, suggesting the exchangeability between His and Asp at this site. DNA binding analysis implicates the involvement of the four residues in metal binding. Mn(2+)-mediated cleavage of inosine-containing DNA is stimulated by the addition of Ca(2+), a metal ion that does not support catalysis. The effects of Mn(2+) on Mg(2+)-mediated DNA cleavage show a complexed initial stimulatory and later inhibitory pattern. The data obtained from the dual metal ion analyses lead to the notion that two metal ions are involved in endonuclease V-mediated catalysis. A catalytic and regulatory two-metal model is proposed.     

Key role of conserved histidines in recombinant mouse beta-carotene 15,15'-monooxygenase-1 activity

Alignment of sequences of vertebrate beta-carotene 15,15'-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His172, His237, His308, His514, Asp52, Glu140, Glu314, Glu405, and Glu457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate non-conserved in the overall family with alanines by site-directed mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V(max). The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase.     

Concerted protonation of key histidines triggers membrane interaction of the diphtheria toxin T domain

The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His(223)-His(257) and His(251) are the most sensitive triggers for the formation of the molten globule state in solution, whereas His(322)-His(323) and His(251) are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.     

Probing electrostatic interactions along the reaction pathway of a glycoside hydrolase: histidine characterization by NMR spectroscopy

We have characterized by NMR spectroscopy the three active site (His80, His85, and His205) and two non-active site (His107 and His114) histidines in the 34 kDa catalytic domain of Cellulomonas fimi xylanase Cex in its apo, noncovalently aza-sugar-inhibited, and trapped glycosyl-enzyme intermediate states. Due to protection from hydrogen exchange, the level of which increased upon inhibition, the labile 1Hdelta1 and 1H epsilon1 atoms of four histidines (t1/2 approximately 0.1-300 s at 30 degrees C and pH approximately 7), as well as the nitrogen-bonded protons in the xylobio-imidazole and -isofagomine inhibitors, could be observed with chemical shifts between 10.2 and 17.6 ppm. The histidine pKa values and neutral tautomeric forms were determined from their pH-dependent 13C epsilon1-1H epsilon1 chemical shifts, combined with multiple-bond 1H delta2/epsilon1-15N delta1/epsilon2 scalar coupling patterns. Remarkably, these pKa values span more than 8 log units such that at the pH optimum of approximately 6 for Cex activity, His107 and His205 are positively charged (pKa > 10.4), His85 is neutral (pKa < 2.8), and both His80 (pKa = 7.9) and His114 (pKa = 8.1) are titrating between charged and neutral states. Furthermore, upon formation of the glycosyl-enzyme intermediate, the pKa value of His80 drops from 7.9 to <2.8, becoming neutral and accepting a hydrogen bond from an exocyclic oxygen of the bound sugar moiety. Changes in the pH-dependent activity of Cex due to mutation of His80 to an alanine confirm the importance of this interaction. The diverse ionization behaviors of the histidine residues are discussed in terms of their structural and functional roles in this model glycoside hydrolase.     

Consequences of molecular engineering enhanced DNA binding in a DNA repair enzyme.

Facilitated one-dimensional diffusion is a general mechanism utilized by several DNA-interactive proteins as they search for their target sites within large domains of nontarget DNA. T4 endonuclease V is a protein which scans DNA in a nonspecifically bound state and processively incises DNA at ultraviolet (UV)-induced pyrimidine dimer sites. An electrostatic contribution to this mechanism of target location has been established. Previous studies indicate that a decrease in the affinity of endonuclease V for nontarget DNA results in a decreased ability to scan DNA and a concomitant decrease in the ability to enhance UV survival in repair-deficient Escherichia coli. This study was designed to question the contrasting effect of an increase in the affinity of endonuclease V for nontarget DNA. With this as a goal, a gradient of increasingly basic amino acid content was created along a proposed endonuclease V-nontarget DNA interface. This incremental increase in positive charge correlated with the stepwise enhancement of nontarget DNA binding, yet inversely correlated with enhanced UV survival in repair-deficient E. coli. Further analysis suggests that the observed reduction in UV survival is consistent with the hypothesis that enhanced nontarget DNA affinity results in reduced pyrimidine dimer-specific recognition and/or binding. The net effect is a reduction in the efficiency of pyrimidine dimer incision.     

Defining amino acid residues involved in DNA-protein interactions and revelation of 3'-exonuclease activity in endonuclease V

Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3' side one nucleotide from a deaminated base lesion. Site-directed mutagenesis analysis was conducted at seven conserved motifs of the thermostable Thermotoga maritima endonuclease V to probe for residues that affect DNA-protein interactions. Y80, G83, and L85 in motif III, H116 and G121 in motif IV, A138 in motif V, and S182 in motif VI affect binding of both the double-stranded inosine-containing DNA substrate and the nicked double-stranded inosine-containing DNA product, resulting in multiple enzymatic turnovers. The substantially reduced DNA cleavage activity observed in G113 in motif IV and G136 in motif V can be partly attributed to their defect in metal cofactor coordination. Alanine substitution at amino acid 118 primarily reduces the level of binding to the nicked product, suggesting that R118 plays a significant role in postcleavage DNA-protein interaction. Binding and cleavage analyses of multiple mutants at positions Y80 and H116 underscore the role these residues play in protein-DNA interaction and implicate their potential involvement as a hydrogen bond donor in recognition of deaminated DNA bases. DNA cleavage analysis using mutants defective in DNA binding reveals a novel 3'-exonuclease activity in endonuclease V. An alternative model is proposed that entails lesion specific cleavage and endonuclease to 3'-exonuclease mode switch by endonuclease V for removal of deaminated base lesions during endonuclease V-mediated repair.     

On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms

The enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP(+)-dependent dehydrogenase reaction or an NADP(+)-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type N(t)-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.     

Bioinformatics and molecular modeling in chemical enzymology. Active sites of hydrolases

Comparison and multiple alignments of amino acid sequences of a representative number of related enzymes demonstrate the existence of certain positions of amino acid residues which are permanently reproducible in all members of the whole family. The use of the bioinformatic approach revealed conservative residues in each of the related enzymes and ranked amino acid conservatism for the overall enzymatic catalysis. Glycine and aspartic acid residues were shown to be the most essential for structure and catalytic activity of enzymes. Amino acid residues forming catalytic subsite of the active site of enzymes are always highly conservative. Analysis revealed that aspartic acid carboxyl group is the most frequently employed nucleophilic (in deprotonated form) and electrophilic (in protonated form) agent involved in activation of molecules by the mechanism of general base and acidic catalyses in the catalytic sites of enzymes. Glycine is a unique amino acid possessing the highest possibilities for rotation along C–C and C–N bonds of the polypeptide chain. The conservative fixation of the glycine residue in polypeptide chains of related enzymes provides a possibility for directed assembly of amino acid residues into the catalytic subsite structure. It is possible that the conservative glycines provide known conformational mobility of the protein and the active site. Methods of molecular modeling were used for analysis of structural substitutions of conservative and non-conservative glycines and their effects on geometry of catalytic site of typical hydrolases. The substitution of glycine(s) for alanine significantly altered the catalytic site structures.     

Site-directed mutagenesis of glutathione S-transferase YaYa: nonessential role of histidine in catalysis

A cDNA encoding a rat liver glutathione S-transferase Ya subunit has been expressed in Escherichia coli and the expressed enzyme purified to homogeneity. In order to examine the catalytic role of histidine in the glutathione S-transferase Ya homodimer, site-directed mutagenesis was used to replace all three histidine residues (at positions 8, 143, and 159) by other amino acid residues. The replacement of histidine 8 or histidine 143 with valine did not affect the 1-chloro-2,4-dinitrobenzene-conjugating activity nor the isomerase activity. However, the replacement of histidine with valine at position 159 produced the mutant GST which exhibited only partial activity. A greater decrease in catalytic activity was observed by histidine----tyrosine or histidine----lysine replacement at position 159. On the other hand, the histidine 159----asparagine mutant retained full catalytic activity. Our results indicate that histidine residues in the Ya homodimer are not essential for catalytic activity. However, histidine 159 might be critical in maintaining the proper conformation of this enzyme since replacement of this amino acid by either lysine or tyrosine did result in significant loss of enzymatic activity.     

Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene

A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.     

Identification of functionally relevant histidine residues in the apoptotic nuclease CAD

The caspase-activated DNase CAD (DFF40/CPAN) degrades chromosomal DNA during apoptosis. Chemical modification with DEPC inactivates the enzyme, suggesting that histidine residues play a decisive role in the catalytic mechanism of this nuclease. Sequence alignment of murine CAD with four homologous apoptotic nucleases reveals four completely (His242, His263, His304 and His308) and two partially (His127 and His313) conserved histidine residues in the catalytic domain of the enzyme. We have changed these residues to asparagine and characterised the variant enzymes with respect to their DNA cleavage activity, structural integrity and oligomeric state. All variants show a decrease in activity compared to the wild-type nuclease as measured by a plasmid DNA cleavage assay. H242N, H263N and H313N exhibit DNA cleavage activities below 5% and H308N displays a drastically altered DNA cleavage pattern compared to wild-type CAD. Whereas all variants but one have the same secondary structure composition and oligomeric state, H242N does not, suggesting that His242 has an important structural role. On the basis of these results, possible roles for His127, His263, His304, His308 and His313 in DNA binding and cleavage are discussed for murine CAD.     

Site-specific mutagenesis of an essential histidine residue in pancreatic cholesterol esterase

The histidine residue essential for the catalytic activity of pancreatic cholesterol esterase (carboxylester lipase) has been identified in this study using sequence comparison and site-specific mutagenesis techniques. In the first approach, comparison of the primary structure of rat pancreatic cholesterol esterase with that of acetylcholinesterase and cholinesterase revealed two conserved histidine residues located at positions 420 and 435. The sequence in the region around histidine 420 is quite different between the three enzymes. However, histidine 435 is located in a 22-amino acid domain that is 47% homologous with other serine esterases. Based on this sequence homology, it was hypothesized that histidine 435 is the histidine residue essential for catalytic activity of cholesterol esterase. The role of His435 in the catalytic activity of pancreatic cholesterol esterase was then studied by the site-specific mutagenesis technique. Substitution of the histidine in position 435 with glutamine, arginine, alanine, serine, or aspartic acid abolished the ability of cholesterol esterase to hydrolyze p-nitrophenyl butyrate and cholesterol [14C]oleate. In contrast, mutagenesis of the histidine residue at position 420 to glutamine had no effect on cholesterol esterase enzyme activity. The results of this study strongly suggested that histidine 435 may be a component of the catalytic triad of pancreatic cholesterol esterase.     

Mutational study of heparan sulfate 2-O-sulfotransferase and chondroitin sulfate 2-O-sulfotransferase

Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated polysaccharides with a wide range of biological functions. Heparan sulfate 2-O-sulfotransferase (HS-2OST) transfers the sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the 2-OH position of the hexauronic acid that is adjacent to N-sulfated glucosamine, whereas chondroitin sulfate 2-O-sulfotransferase (CS-2OST) transfers the sulfo group to the hexauronic acid that is adjacent to N-acetylated galactosamine. Here we report a systematic mutagenesis study of HS-2OST and CS-2OST based on their structural homology to estrogen sulfotransferase and HS 3-O-sulfotransferase isoform 3 (3-OST3), for which crystal structures exist. We have identified six residues possibly involved in binding to PAPS. HS-2OST carrying mutations of these residues lacks sulfotransferase activity and the ability to bind 3'-phosphoadenosine 5'-phosphate, a PAPS analogue, as determined by isothermal titration calorimetry. Similar residues involved in binding to PAPS were also identified in CS-2OST. Additional residues that participate in carbohydrate substrate binding were also identified in both enzymes. Mutations at these residues led to the loss of sulfotransferase activity but maintained the ability to bind to phosphoadenosine 5'-phosphate. The catalytic function of HS-2OST appears to involve two histidine residues (His140 and His142), whereas only one histidine (His168) of CS 2-OST is likely to be critical. This unique feature of HS 2-OST catalytic residues directed us to characterize the Drosophila heparan sulfate 2-O-sulfotransferase. The results from this study provide insight into the differences and similarities various residues play in the biological roles of the HS-2OST and CS-2OST enzymes.     

Photocross-linking of the homing endonuclease PI-SceI to its recognition sequence

PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family of homing endonucleases. According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding. To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA. Sixty-three double-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm). The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand. The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction. Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI. This inactivation can be suppressed by substrate binding. This result further supports the finding that at least one His residue is in close contact to the DNA. Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI.     

Non-essentiality of cysteine and histidine residues for the activity of human class PI glutathione S-transferase.

In order to examine the roles of cysteine and histidine residues in the activity of human class Pi glutathione S-transferase (GST pi), site-directed mutagenesis was used to replace each of the four cysteine residues (at positions 14, 47, 101 and 169) with serine and each of the two histidine residues (at positions 71 and 162) with asparagine using a cDNA for the enzyme (Kano, T. et al. (1987) Cancer Res., 47, 5626-5630) and an E. coli expression system. The replacements of Cys101, Cys169, His71 and His162 did not affect the GSH-conjugating activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid. On the other hand, the activities were partly decreased by the replacements of Cys47 and Cys14. These results indicated that the cysteine and histidine residues in GST pi are not essential for the catalytic activity, although Cys47 and Cys14 may contribute to some extent to the catalytic efficiency.     

Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding

In order to turn the subunit association and biotin binding of avidin into pH-sensitive phenomena, we have replaced individually three amino acid residues in avidin (Met96, Val115 and Ile117) with histidines in the 1-3 interface, and in combination with a histidine conversion in the 1-2 interface (Trp110). The single replacements Met96His and Val115His in the 1-3 interface were found to have a clear effect on the quaternary structure of avidin, since subunit associations of these mutants became pH-dependent. The histidine replacement in the 1-2 interface affected the biotin-binding properties of the mutants, in particular reversibility of binding and protein-ligand complex formation were pH-sensitive, as measured by IAsys biosensor and fluorescence correlation spectroscopy, respectively. The possibility of regulating the quaternary structure and function of avidin in a controlled and predictable manner, due to introduced interface histidines, will expand even further the range and versatility of the avidin-biotin technology.