Mutagenesis of the three conserved valine residues: consequence on the foldability of insulin

Natural polypeptide chain usually can spontaneously fold into tightly compact native structure. This capability is the so-called foldability. However, how the foldability is encoded in the polypeptide chain is still poorly understood. The structure of insulin has been well solved and extensively investigated. Therefore, insulin provides a good model for investigating the role of individual residue to the sequence foldability. In insulins from different species there are three highly conserved Val residues (A3Val, B12Val, and B18Val), but their contribution to the insulin foldability is still unknown. Here, a single-chain insulin (PIP) was used to investigate the contribution of the three conserved valine residues to the foldability. Five PIP mutants, [A3S]PIP, [A3T]PIP, [B12A]PIP, [B18T]PIP, and [B18L]PIP, were used in the studies, and their structural changes, secretion efficiency, structural stability, disulfide stability, and in vitro refolding efficiency were analyzed. The effects of the mutations on the PIP foldability are multifold: as a whole, mutation of A3Val has only moderate effect; while mutation of B12Val has significant detriment; hydrophobic replacement of B18Val is more tolerant than hydrophilic substitution as foldability is concerned. Therefore, the three highly conserved valine residues have different contributions to the insulin foldability, and their contribution might be ranked as B12Val>B18Val>A3Val.     

Mutation of conserved tryptophan residues at the dimer interface of Staphylococcus aureus nitric oxide synthase

Nitric oxide synthases (NOSs) share two invariant tryptophan residues within a conserved helical lariat that is part of the pterin-binding site and dimer interface. We mutated Staphylococcus aureus NOS Trp-314 (to alanine, phenylalanine, tyrosine and histidine) and Trp-316 (to alanine, phenylalanine and tyrosine) and characterized the effects of mutation on heme environment, quaternary structure, enzymatic activity, and substrate affinity. With arginine present, all saNOS variants bound heme with native thiolate ligation, formed high spin ferric complexes and were dimeric. All variants catalyze the peroxide-dependent oxidation of N-hydroxy-l-arginine, at rates from 10% to 55% of wild type activity. Arginine-free proteins are dimeric with the exception of W314A. Arginine affinity for all variants decreases with increasing temperature between 15 and 42 °C but is precipitous for position-314 variants. Previous structural and biophysical characterization of NOS oxygenase domains demonstrated that the protein can exist in either a tight or loose conformation, with the former corresponding to the active state of the protein. In the position-314 variants it is likely that the loose conformation is favoured, owing to the loss of a hydrogen bond between the indole side chain and the polypeptide backbone of the helical lariat.     

Modification of tryptophan and methionine residues is implicated in the oxidative inactivation of surfactant protein B

Exposing BLES (bovine lipid extract surfactant), a clinical surfactant, to reactive oxygen species (ROS) alters surfactant protein B (SP-B), as indicated by Coomassie Blue staining, silver staining, and Western analysis. Hypochlorous acid (HOCl) treatment leads to elevated maximum surface tension (gammamax) and a deterioration in minimum gamma (gammamin) during surface area cycling. Fenton reaction resulted in immediate increases in gammamin and gammamax. Intrinsic fluorescence measurements indicated Fenton, but not HOCl, induced conversion of Trp9 of SP-B to hydroxyTrp (OHTrp), N-formylkynurenine (NFKyn), and kynurenine (Kyn). Electrospray ionization mass spectrometry (ESI-MS) revealed molecular weight alterations consistent with oxidation of Met (HOCl, Fenton) and Trp (Fenton) residues. Oxidative alterations to Met29 and Met65 (HOCl, Fenton) and to Trp9 (OHTrp with HOCL and NFKyn plus Kyn with Fenton) were confirmed by matrix-assisted laser desorption mass spectrometry (MALDI-MS) studies on SP-B tryptic fragments. Some Met oxidation was observed with control SP-B. When taken together with captive bubble tensiometer measurements, these studies suggest that Met oxidation of SP-B by HOCl or Fenton interferes with phospholipid respreading during compression-expansion of surfactant films, while Fenton oxidation, which produces more extensive Met oxidation and disruption of the indole ring of Trp9, further abrogated the ability of such films to attain low surface tensions during compression. These studies provide insight into the manner by which ROS generated during acute lung injury and the acute respiratory distress syndrome act to inhibit not only endogenous surfactant but also therapeutic surfactants administered to counteract these conditions.     

Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.

We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor.     

The conversion of phosphoserine residues to selenocysteine residues on an opal suppressor tRNA and casein

This study has been undertaken in order to elucidate the mechanisms of incorporation of Se into glutathione peroxidase (GSHPx), in which selenocysteine corresponds to the opal termination codon UGA on the mRNA. We studied the above mechanisms using an opal suppressor tRNA, prepared from bovine liver, and casein as a model protein for the GSHPx apo-enzyme which might contain phosphoserine. The results showed that opal suppressor tRNA did not accept selenocysteine (lower than 0.1 mmol/mol) under the standard conditions. A trace amount of phosphoseryl-tRNA was converted to selenocysteyl-tRNA by incubation with H2Se and some enzymes. Meanwhile, a number of phosphoserine residues in casein were converted to selenocysteine residues by incubation with H2Se and enzymes. These results suggest that opal suppressor tRNA plays a role in synthesizing GSHPx via co- and/or post-translational mechanisms.     

Two conserved tryptophan residues of tumor necrosis factor and lymphotoxin are not involved in the biological activity

Each of the two highly conserved tryptophan residues in hTNF (positions 28 and 114) was converted into phenylalanine by site-directed mutagenesis and the mutant proteins were partially purified. A cytotoxicity assay on mouse L929 cells showed only a slight reduction in biological activity, strongly suggesting that neither of the two amino acids is involved in the active site.     

The role of conserved tryptophan and acidic residues in the human dopamine transporter as characterized by site-directed mutagenesis

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na(+)/Cl(-)-dependent transporters. We have explored the roles of these residues using non-conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10-24-fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [(3)H]DA uptake, but caused undetectable [(3)H]CFT binding. Upon anti-C-terminal-hDAT immunoblotting, D345N appeared as broad bands of 66-97 kDa, but this band could not be photoaffinity labeled with cocaine analog [(125)I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid ([(125)I]RTI-82). Unexpectedly, in this mutant, cocaine-like drugs remained potent inhibitors of [(3)H]DA uptake. CFT solely raised the K(m) of [(3)H]DA uptake in wild-type hDAT, but increased K(m) and decreased V(max) in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.     

Chemical modification of two tryptophan residues abolishes the catalytic activity of aminoacylase.

1) The reaction of 1 H-diazotetrazole and N-bromosuccinimide with aminoacylase was studied under different conditions. A tenfold molar excess of 1 H-diazotetrazole (2 X 10(-4) M) at pH 5.5 abolishes the catalytic activity of the enzyme while modifying only two tryptophan residues. No other amino acid reacted under these conditions as tested by amino acid analysis. 2) With a 40-fold molar excess of N-bromosuccinimide (8 X 10(-4)M) at pH 5.0, two tryptophan residues of the enzyme were oxidized with complete loss of activity. Under these conditions no significant cleavage of the polypeptide chain was observed. Neither tyrosine nor histidine was modified by this reagent, up to a 100-fold molar excess. 3) Substrates and reversible (N-tosylalanine) and irreversible (TosPheCH2Cl) inhibitors of the enzyme do not protect the two reactive tryptophans against the modification reagents. Under more drastic conditions, lysine, tyrosine and histidine residues are also modified by the reagents.     

Autoreduction of ferryl myoglobin: discrimination among the three tyrosine and two tryptophan residues as electron donors

Ferric myoglobin undergoes a two-electron oxidation in its reaction with H(2)O(2). One oxidation equivalent is used to oxidize Fe(III) to the Fe(IV) ferryl species, while the second is associated with a protein radical but is rapidly dissipated. The ferryl species is then slowly reduced back to the ferric state by unknown mechanisms. To clarify this process, the formation and stability of the ferryl forms of the Tyr --> Phe and Trp --> Phe mutants of recombinant sperm whale myoglobin (SwMb) were investigated. Kinetic studies showed that all the mutants react normally with H(2)O(2) to give the ferryl species. However, the rapid phase of ferryl autoreduction typical of wild-type SwMb was absent in the triple Tyr --> Phe mutant and considerably reduced in the Y103F and Y151F mutants, strongly implicating these two residues as intramolecular electron donors. Replacement of Tyr146, Trp7, or Trp14 did not significantly alter the autoreduction, indicating that these residues do not contribute to ferryl reduction despite the fact that Tyr146 is closer to the iron than Tyr151 or Tyr103. Furthermore, analysis of the fast phase of autoreduction in the dimer versus recovered monomer of the Tyr --> Phe mutant K102Q/Y103F/Y146F indicates that the Tyr151-Tyr151 cross-link is a particularly effective electron donor. The presence of an additional, slow phase of reduction in the triple Tyr --> Phe mutant indicates that alternative but normally minor electron-transfer pathways exist in SwMb. These results demonstrate that internal electron transfer is governed as much by the tyrosine pK(a) and oxidation potential as by its distance from the electron accepting iron atom.     

Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat.

The likelihood for improvement in the catalytic properties of Escherichia coli alkaline phosphatase was examined using site-directed mutagenesis. Mutants were constructed by introducing sequence changes into nine preselected amino acid sites within 10 A of the catalytic residue serine 102. When highly conserved residues in the family of alkaline phosphatases were mutated, many of the resulting enzymes not only maintained activity, but also exhibited greatly improved kcat. Of approximately 170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity. In particular, a substitution by serine of a totally invariant Asp101 resulted in a 35-fold increase of specific activity over wild-type at pH 10.0. Up to 6-fold increases of the kcat/Km ratio were observed.     

Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile. Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide. The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose. A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised. For the wild-type enzyme, the value of the Br?nsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups. Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining. The Br?nsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively). These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1). Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism.     

Mutational analysis of the role of tryptophan residues in an antimicrobial peptide

Antimicrobial peptides belonging to the pediocin-like family of bacteriocins (class IIa bacteriocins) produced by lactic acid bacteria contain several tryptophan residues that are highly conserved. Since tryptophan residues in membrane proteins are often positioned in the membrane-water interface, we hypothesized that Trp residues in bacteriocins could be important determinants of the structure of membrane-bound peptides and of anti-microbial activity. To test this hypothesis, the effects of mutating each of the 3 tryptophan residues (Trp18, Trp33, and Trp41) in the 43-residue pediocin-like bacteriocin sakacin P were studied. Trp18 and Trp33 are located at each end of an amphihilic alpha-helix, whereas Trp41 is near the end of an unstructured C-terminal tail. Replacement of Trp33 with the hydrophobic residues Leu and Phe had marginal effects on activity, whereas replacement with the more polar Tyr and Arg reduced activity 10-20 and 500-1000 times, respectively, indicating that Trp33 and the C-terminal part of the helix interact with the hydrophobic core of the membrane. Any mutation of Trp18 and Trp41 reduced activity, indicating that these two residues play unique roles. Substitutions with other aromatic residues were the least deleterious, indicating that both Trp18 and Trp41 interact with the membrane-water interface. The suggested locations of the three Trp residues are compatible with a structural model in which the helix and the C-terminal tail form a hairpin-like structure, bringing Trp18 and Trp41 close to each other in the interface, and placing Trp33 in the hydrophobic core of the membrane. Indeed, the deleterious effect of the W18L and W41L mutations could be overcome by stabilizing the hairpin-like structure by introduction of a disulfide bridge between residues 24 and 44. These results provide a basis for a refined structural model of pediocin-like bacteriocins and highlight the unique role that tryptophan residues can play in membrane-interacting peptides.     

Characterization of the AP endonucleases from Thermoplasma volcanium and Lactobacillus plantarum: Contributions of two important tryptophan residues to AP site recognition

Escherichia coli AP endonuclease (ExoIII) and its human homolog (APE1) have the sole tryptophan residue for AP site recognition (AP site recognizer) but these residues are at different positions near the catalytic sites. On the other hand, many bacterial AP endonucleases have two tryptophan residues at the same positions of both ExoIII and APE1. To elucidate whether these residues are involved in AP site recognition, the ExoIII homologs of Thermoplasma volcanium and Lactobacillus plantarum were characterized. These proteins showed AP endonuclease and 3'-5'exonculease activities. In each enzyme, the mutations of the tryptophan residues corresponding to Trp-280 of APE1 caused more significant reductions in activities and binding abilities to the oligonucleotide containing an AP site (AP-DNA) than those corresponding to Trp-212 of ExoIII. These results suggest that the tryptophan residue corresponding to Trp-280 of APE1 is the predominant AP site recognizer, and that corresponding to Trp-212 of ExoIII is the auxiliary recognizer.     

Role of conserved proline residues in stabilizing tryptophan synthase alpha subunit: analysis by mutants with alanine or glycine

To study the role of Pro residues in the conformation and conformational stability of a protein, nine mutant alpha subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six Pro residues (positions 28, 57, 62, 96, 132, and 207) that are conserved in 10 microorganisms, were constructed by means of site-directed mutagenesis. The far-ultraviolet (UV) CD spectra of five mutant alpha subunits with Ala in place of Pro were identical to the spectrum of the wild-type protein, the exception being the mutant at position 207 (P207A). CD values in the far-UV region were less negative for P207A, indicating that the Pro residue at position 207 plays a role in maintaining the intact structure of the alpha subunit. The negative CD values of the Gly mutants examined (P28G, P96G, and P132G) were also decreased. Calorimetric measurements showed that the two mutants at position 28 (P28G and P28A) gave two peaks in the excess heat capacity curve, whereas the wild type and other Pro mutants had only a single peak. The stability of each mutant protein relative to that of the wild type was about the same for P57A, less for P62A and P132A, and markedly decreased for P96A and P207A, which are substituted at less mobile positions. The changes of denaturation entropy (delta delta dS) at the denaturation temperature of the wild-type protein (54.1 degrees C at pH 9.0) were positive for P57A, P62A, and P132A, but negative for P96A, P207A, and P132G.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of tryptophan residues in an integral membrane protein: diacylglycerol kinase

Tryptophan residues are thought to play special roles in integral membrane proteins, anchoring transmembrane alpha-helices into the lipid bilayer. We have studied the effect of mutating the five Trp residues in the diacylglycerol kinase (DGK) of Escherichia coli to Leu residues. The fluorescence emission maxima for DGK and a variety of Trp mutants in bilayers of dioleoylphosphatidylcholine [di(C18:1)PC] are all centered at ca. 327 nm, suggesting that all five Trp residues are located close to the glycerol backbone region of the bilayer. This is also consistent with fluorescence quenching experiments, measuring the separation between the Trp residues and the bromine atoms in a bilayer of dibromostearoylphosphatidylcholine. Mutation of Trp residues in DGK was found to have significant effects on activity for DGK reconstituted into bilayers of di(C18:1)PC containing 30 mol % 1,2-dihexanoylglycerol (DHG). Of the mutants containing a single Trp residue, only that containing Trp-112 was found to give active protein. The presence of both Trp-25 and Trp-112 gave higher activity than Trp-112 alone. Trp-25 and Trp-112 are the most important Trp residues in DGK as far as activity is concerned. Effects of mutations on K(m) for DHG were generally greater than effects on v(max). The activity of wild-type and mutant DHGs reconstituted into bilayers of phosphatidylcholines was sensitive to the chain length of the phospholipid, with highest activities for chain lengths of C18 or C20 and lower activities in phosphatidylcholines with shorter or longer chains. Compared to wild-type DGK, the Trp mutants were less affected by long-chain phosphatidylcholines but more affected by short-chain phospholipids. In mutants lacking Trp-25, low activities in short-chain phospholipids followed from a decrease in v(max) compared to wild type, combined with an increase in K(m) value for DHG, as observed in the wild type. It is suggested that Trp-25 plays a role in maintaining the alignment of ATP and DHG at the active site. Fluorescence emission spectra for the Trp mutants do not change significantly with changing fatty acyl chain length from C14 to C24, showing efficient hydrophobic matching between DGK and the surrounding lipid bilayer. It is suggested that hydrophobic matching is achieved by tilting of the transmembrane alpha-helix or rotation of residues at the ends of the helices about the Calpha-Cbeta bond linking the residue to the helix backbone. As well as any structural effects, the presence of Trp residues in DGK has a clear effect on thermal stability.     

Mutagenesis of two acidic active site residues in human muscle creatine kinase: implications for the catalytic mechanism

Creatine kinase (CK) catalyzes the reversible phosphorylation of the guanidine substrate, creatine, by MgATP. Although several X-ray crystal structures of various isoforms of creatine kinase have been published, the detailed catalytic mechanism remains unresolved. A crystal structure of the CK homologue, arginine kinase (AK), complexed with the transition-state analogue (arginine-nitrate-ADP), has revealed two carboxylate amino acid residues (Glu225 and Glu314) within 2.8 A of the proposed transphosphorylation site. These two residues are the putative catalytic groups that may promote nucleophilic attack by the guanidine amino group on the gamma-phosphate of ATP. From primary sequence alignments of arginine kinases and creatine kinases, we have identified two homologous creatine kinase acidic amino acid residues (Glu232 and Asp326), and these were targeted for examination of their potential roles in the CK mechanism. Using site-directed mutagenesis, we have made several substitutions at these two positions. The results indicate that of these two residues the Glu232 is the likely catalytic residue while Asp326 likely performs a role in properly aligning substrates for catalysis.     

Sensitivity of P-glycoprotein tryptophan residues to benzodiazepines and ATP interaction

Plasma membrane P-glycoprotein is a member of the ATP-binding cassette family of membrane transporters. In the present study tryptophan intrinsic fluorescence was used to understand the P-glycoprotein response to three benzodiazepines (bromazepam, chlordiazepoxide and flurazepam) in the presence and absence of ATP. Fluorescence emission spectra showed a red shift on the maximal emission wavelength upon interaction of P-glycoprotein with all benzodiazepines. Benzodiazepine association with nucleotide-bound P-glycoprotein also showed this trend and the quenching profile was attributed to a sphere-of-action model, for static fluorescence. Furthermore, quenching data of benzodiazepine-bound P-glycoprotein with ATP were concentration dependent and saturable, indicating that nucleotide binds to P-glycoprotein whether drug is present or not. These results seems in agreement with the proposal of the ATP-switch model by Higgins and Linton, where substrate binding to the transporters initiates the transport cycle by increasing the ATP binding affinity.     

Targeted mutagenesis of five conserved tryptophan residues of LolB involved in membrane localization of Escherichia coli lipoproteins

LolB, catalyzing the last step of lipoprotein transfer from the inner to the outer membrane of Escherichia coli, is itself a lipoprotein anchored to the outer membrane. Five Trp residues of LolB are conserved among LolB homologs in Gram-negative bacteria. These Trp residues were mutagenized to obtain defective LolB mutants. Mutation of Trp at position 52 to Pro impaired the receptor activity and caused accumulation of the LolA-lipoprotein complex in the periplasm. Similar mutants were obtained for Trp at position 117. A mutant with Gly instead of Trp at position 148 retained the receptor activity but inhibited growth upon its overproduction. The outer membrane sorting of this mutant seemed to be defective, lipoprotein transfer thereby being perturbed when it was overproduced. Despite the strong conservation, no defective mutant for Trp at position 183 was obtained, and only weak mutants were isolated for Trp at position 18. Based on the crystal structure of LolB, the phenotypes of these mutants are discussed.     

Roles of two conserved cysteine residues in the activation of human adenovirus proteinase.

The roles of two conserved cysteine residues involved in the activation of the adenovirus proteinase (AVP) were investigated. AVP requires two cofactors for maximal activity, the 11-amino acid peptide pVIc (GVQSLKRRRCF) and the viral DNA. In the AVP-pVIc crystal structure, conserved Cys104 of AVP has formed a disulfide bond with conserved Cys10 of pVIc. In this work, pVIc formed a homodimer via disulfide bond formation with a second-order rate constant of 0.12 M(-1) s(-1), and half of the homodimer could covalently bind to AVP via thiol-disulfide exchange. Alternatively, monomeric pVIc could form a disulfide bond with AVP via oxidation. Regardless of the mechanism by which AVP becomes covalently bound to pVIc, the kinetic constants for substrate hydrolysis were the same. The equilibrium dissociation constant, K(d), for the reversible binding of pVIc to AVP was 4.4 microM. The K(d) for the binding of the mutant C10A-pVIc was at least 100-fold higher. Surprisingly, the K(d) for the binding of the C10A-pVIc mutant to AVP decreased at least 60-fold, to 6.93 microM, in the presence of 12mer ssDNA. Furthermore, once the mutant C10A-pVIc was bound to an AVP-DNA complex, the macroscopic kinetic constants for substrate hydrolysis were the same as those exhibited by wild-type pVIc. Although the cysteine in pVIc is important in the binding of pVIc to AVP, formation of a disulfide bond between pVIc and AVP was not required for maximal stimulation of enzyme activity by pVIc.     

Chemical modification of lysine residues in Bacillus licheniformis alpha-amylase: conversion of an endo- to an exo-type enzyme

The lysine residues of Bacillus licheniformis alpha-amylase (BLA) were chemically modified using citraconic anhydride or succinic anhydride. Modification caused fundamental changes in the enzymes specificity, as indicated by a dramatic increase in maltosidase and a reduction in amylase activity. These changes in substrate specificity were found to coincide with a change in the cleavage pattern of the substrates and with a conversion of the native endo- form of the enzyme to a modified exo- form. Progressive increases in the productions of rho-nitrophenol or glucose, when para nitrophenyl-maltoheptaoside or soluble starch, respectively, was used as substrate, were observed upon modification. The described changes were affected by the size of incorporated modified reagent: citraconic anhydride was more effective than succinic anhydride. Reasons for the observed changes are discussed and reasons for the effectivenesses of chemical modifications for tailoring enzyme specificities are suggested.