Modification of arginine residues in calf intestinal alkaline phosphatase

Calf intestinal alkaline phosphatase is inactivated by 2,3-butanedione and phenylglyoxal. The reaction with either reagent results in a biphasic loss of enzymatic activity. Inactivation by 2,3-butanedione in borate buffer can be reversed after gel-filtration in Tris buffer but no enzyme reactivation is observed after phenylglyoxal treatment. Phosphate, ATP and NADH protect the enzyme from both compounds while no protection is displayed by L-phenylalanine. The selective chemical modification indicates that two differently reacting types of arginines are present in the active site domains of the dimeric enzyme.     

Identification of histidine residues at the active site of Megalobatrachus japonicus alkaline phosphatase by chemical modification

Alkaline phosphatase from Megalobatrachus japonicus was inactivated by diethyl pyrocarbonate (DEP). The inactivation followed pseudo-first-order kinetics with a second-order rate constant of 176 M(-1) x min(-1) at pH 6.2 and 25 degrees C. The loss of enzyme activity was accompanied with an increase in absorbance at 242 nm and the inactivated enzyme was re-activated by hydroxylamine, indicating the modification of histidine residues. This conclusion was also confirmed by the pH profiles of inactivation, which showed the involvement of a residue with pK(a) of 6.6. The presence of glycerol 3-phosphate, AMP and phosphate protected the enzyme against inactivation. The results revealed that the histidine residues modified by DEP were located at the active site. Spectrophotometric quantification of modified residues showed that modification of two histidine residues per active site led to complete inactivation, but kinetic stoichiometry indicated that one molecule of modifier reacted with one active site during inactivation, probably suggesting that two essential histidine residues per active site are necessary for complete activity whereas modification of a single histidine residue per active site is enough to result in inactivation.     

Studies on alkaline phosphatase. Phosphorylation of calf intestinal alkaline phosphatase by 32P-labelled pyrophosphate

1. A purified preparation of alkaline phosphatase from calf-intestinal mucosa was phosphorylated by (32)P-labelled PP(i) at a serine residue on the enzyme. Under the conditions employed, up to 0.15mum-labelled sites were obtained from 1mum-[(32)P]PP(i). 2. The phosphorylated enzyme was labile, the rate of dephosphorylation being similar to the overall rate of substrate hydrolysis. 3. A stopped-flow technique was used to determine the number of phosphomonoesterase active sites, which agreed with the number of (32)P-labelled sites. 4. It is concluded that calf-intestinal alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase.     

Kinetic study on chemical modification of Taka-amylase A. II. Ethoxycarbonylation of histidine residues

The modification of Taka-amylase A (TAA) [EC 3.2.1.1] of Aspergillus oryzae by diethylpyrocarbonate (DEP) was carried out at 25 degrees C and at pH 5.8 (0.1 M acetate buffer). Two out of the six histidine residues were modified with 4.6 mM DEP, and two or three histidine residues were modified with 23 mM DEP. In both cases, one of them was protected from modification by the presence of 15% maltose. The results suggest that two or three out of the six histidine residues are exposed on the surface of the TAA molecule, and one of them exists near the maltose binding site. Ethoxycarbonylation of histidine residues of TAA caused loss of the amylase activity and activation of the hydrolysis of phenyl alpha-maltoside (phi alpha M). The kinetic parameters of the modified TAA for several substrates and analogs were determined at 25 degrees C and at pH 5.3 (0.08 M acetate buffer). From the results, it was found that this alteration of the enzyme activity by the modification was not due to a change in Km value but to a change in k0 value. Thus, some of the histidine residues in TAA are suggested to play an important role in the enzyme catalytic function.     

Inactivation of intestinal alkaline phosphatase by inositol hexaphosphate-Cu (II) coordinate complexes

Alkaline phosphatase (APase) was greater than 99% inactivated upon incubation with myo-inositol hexakisphosphate (IHP) and Cu(II) ions. In the absence of Cu(II), IHP did not inactivate the enzyme. Likewise, cupric ions alone did not produce inactivation. Reactions of APase with IHP plus Cu(II) were competitively inhibited by zinc ions. In contrast to the marked effect of (IHP-Cu) chelate complexes on APase activity, the complexes of IHP with either Zn(II) or Mn(II) had no discernable effect. Both the extent and the rate of activity loss were dependent on the combined IHP and Cu(II) concentration. At an IHP to Cu(II) ratio of 11.6, the extent of inactivation was approximately proportional to the Cu(II) concentration with maximal inactivation attained above 10 microM. Under the same conditions, a nonlinear relation (saturation kinetics) was observed between the pseudo first-order rate constants for the reaction and the IHP and Cu(II) concentration. On the basis of adherence of the data to a mechanism involving an intermediate whose concentration was rate determining, it was suggested that a ternary complexes composed of the apoprotein, the catalytic site zinc ions, and one or more specific IHP-Cu(II) complex [( IHP-Cu]*) may be the first step along the reaction coordinate. Relevant to this possibility which assumes active site interaction is the fact that both IHP alone and (IHP-Cu) complexes are good competitive inhibitors of p-nitrophenyl phosphate hydrolysis under the same solution conditions wherein APase inactivation occurs in the absence of substrate. Rates of enzyme inactivation are decreased with an increase in pH from 6.5 to 8.0. They are also dependent upon buffer type and concentration, apparently related to their association constants for cupric ion binding. Over and above such specific effects, rates of inactivation are also reduced with an increase in ionic strength. Depending on the ratio and concentrations of IHP and Cu(II) used in the reaction with APase, subsequent exposure to EDTA followed by assay in the presence of Zn(II) gave recoveries of activity ranging from 60% to 100%. Both the prior inactivated enzyme (containing IHP and cupric ions) in the presence of EDTA and the native APase upon simultaneous exposure to IHP, Cu(II), and EDTA were slowly and irreversibly inactivated. Correction for this effect gave reconstitution of activity of the (IHP-Cu)-inactivated APase by Zn(II) addition equivalent to that which could be obtained by EDTA-treatment of the native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase. 31P and 113Cd NMR

Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase. This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species. The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II]B)(Cd(II)ACd(II)B) alkaline phosphatase. 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions. From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation. A site metal also serves to coordinate phosphate in the E X P complex. B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site. Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium. There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site.     

The role of tryptophanyl residues in heavy meromyosin as studied by chemical modification with 2-hydroxy-5-nitrobenzyl bromide.

1. Two moles of 2-hydroxy-5-nitrobenzyl group bound selectively to one mole of heavy meromyosin when it was treated with 2-hydroxy-5-nitrobenzyl bromide, a specific reagent for tryptophanyl residues. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum with and without ADP of the bound 2-hydroxy-5-nitrobenzyl groups were measured with heavy meromyosin modified with various amounts of reagent. The properties of the modified heavy meromyosin did not change until the molar binding ratio of the reagent, rH, was about 1, but the properties changed remarkably when rH increased from 1 to 2. 2. Subfragment-1 was prepared from the modified heavy meromyosin by trypsin [EC 3.4.21.4] digestion. The molar binding ratio of the reagent in subfragment-1, rS, was found to be less than 0.1 when rH of the starting heavy meromyosin was less than 0.8. However, rS was about 0.5 in subfragment-1 prepared from heavy meromyosin of rH about 2. The results indicate that only one mole of 2-hydroxy-5-nitrobenzyl group, which was bound with lower reactivity than the other, was bound to a head part of heavy meromyosin. 3. Subfragment-1 fraction prepared from the modified heavy meromyosin could be separated into two fractions by DE-32 cellulose column chromatography; the subfragment-1 portion which eluted later showed a higher rS than that eluted in front. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum induced by ATP were measured with the modified subfragment-1 separated by DE-32 cellulose column chromatography. The ADP-binding ability and the size of the initial burst were not dependent on rS, and coincided with those of subfragment-1 prepared from unmodified heavy meromyosin. 4. The results of ADP binding studies suggest that heavy meromyosin is constituted from nonidentical subunits, and that there is an interaction between them which controls the ADP binding. Two tryptophanyl residues having specific reactivity toward 2-hydroxy-5-nitrobenzyl bromide are assumed to be involved in the interaction.     

Conformational changes and inactivation of calf intestinal alkaline phosphatase in trifluoroethanol solutions

The changes in activity and unfolding of calf intestinal alkaline phosphatase (CIP) during denaturation in different concentrations of trifluoroethanol (TFE) have been investigated by far-ultraviolet circular dichroism and fluorescence emission spectra. Unfolding and activation rate constants were measured and compared, the activation and inactivation courses were much faster than that of unfolding, which suggests that the active site of CIP containing two zinc ions and one magnesium ion is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole. However, compared to other metalloenzymes, CIP is inactivated at higher concentrations of TFE as denaturant.     

Effect of Zn(II) and Mg(II) on phosphohydrolytic activity of rat matrix-induced alkaline phosphatase

Rat matrix-induced alkaline phosphatase is an enzyme which requires magnesium and zinc ions for its maximal activity. Two Zn(II) ions and one Mg(II) ion are bound to each subunit of native dimeric enzyme. The presence of magnesium ion (10-100 microM) or zinc ion (7-20 nM) alone is sufficient to stimulate apoenzyme activity. However maximal activity (264 U/mg) requires the presence of both ions. Binding of Zn(II) ions to the Mg(II) binding site causes a strong inhibition of the apoenzyme while the binding of Mg(II) on Zn(II) binding site is not sufficient to stimulate PNPPase activity of the apoenzyme. Binding of both ions to the enzyme molecule did not change the apparent dissociation constant for PNPP hydrolysis.     

Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.     

The unfolding intermediate state of calf intestinal alkaline phosphatase during denaturation in guanidine solutions

The equilibrium unfolding of calf intestinal alkaline phosphatase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and ultraviolet difference spectra. At low concentrations of GdmCl (< 1.6 M), the fluorescence intensity decreased with a slight red shift of the emission maximum from 332 nm to 344 nm. An unfolding intermediate state was observed at a broad concentration range of GdmCl as a denaturant (between 1.6 and 2.6 M). This intermediate was characterized by increased fluorescence emission intensity, ultraviolet difference absorption at 236 nm and 260 nm, as well as increased binding to the protein and red shift of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid.     

Reversible inhibition of intestinal alkaline phosphatase by inositol hexaphosphate and its Cu(II) coordinate complexes

The influence of inositol hexakisphosphate (IHP) and its cupric ion chelate complexes on alkaline phosphatase (APase) catalysis of p-nitrophenyl phosphate hydrolysis at pH 7.2 has been determined. Both IHP and (IHP-Cu) complexes, but not Cu(II) alone, are effective inhibitors of the enzyme and are of the strictly competitive type with Ki values in the microM range. Without added inhibitors present, the kinetic parameters are kcat 5.7 x 10(3) min(-1); and KM, 18 microM. In the presence of 62 microM IHP, kcat was essentially unchanged with an apparent KM of 68 microM giving a Ki of 22 microM. In the presence of an (IHP-Cu) complex (62 microM IHP, 128 microM Cu(II], the apparent KM was 55 microM and Ki was 30 microM. At a ratio of Cu(II):IHP of 6.0 (372:62 microM) the apparent KM was 30 microM and Ki was 94 microM. The inhibitory effect of (IHP-Cu) complexes thus decreases as the IHP binding sites for cupric ions become saturated. A high ionic strength environment markedly reduces the inhibitory effect of IHP. Previous studies have also shown that rates of APase inactivation by (IHP-Cu) complexes are also ionic strength sensitive [1]. The inhibition of APase activity by either IHP or its coordinate complexes with cupric ions is evidence for their interaction at the enzyme's catalytic sites. Such results thus provide support for an essential element of the mechanism previously suggested for the reversible inactivation (as opposed to inhibition) of APase by (IHP-Cu) chelate complexes, viz., that it may be due to a metal ion exchange reaction leading to the formation of a Cu(II)-substituted enzyme.     

Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification.

The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the beta-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604-607], each of the exposed tryptophans interacts reversibly with cellulose.     

Zn2+ binding to human calbindin D(28k) and the role of histidine residues

We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.     

Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues

Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.