Identification of histidine residues that act as zinc ligands in beta-lactamase II by differential tritium exchange.

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified.     

Histidine residues regulate the transition of photoexcited rhodopsin to its active conformation, metarhodopsin II.

The biologically active photoproduct of rhodopsin, metarhodopsin II (M II), exists in a pH-sensitive equilibrium with its precursor, metarhodopsin I (M I). Increasing acidity favors M II, with the midpoint of the pH titration curve at pH 6.4. To test the long-standing proposal that histidine protonation regulates this conformational transition, we characterized mutant rhodopsins in which each of the 6 histidines was replaced by phenylalanine or cysteine. Only mutants substituted at the 3 conserved histidines showed abnormal M I-M II equilibria. Those in which His-211 was replaced by phenylalanine or cysteine formed little or no M II at either extreme of pH, whereas mutants substituted at His-65 or at His-152 showed enhanced sensitivity to protons. The simplest interpretation of these results is that His-211 is the site where protonation strongly stabilizes the M II conformation and that His-65 and His-152 are sites where protonation modestly destabilizes the M II conformation.     

Identification of residues in beta-lactamase critical for binding beta-lactamase inhibitory protein

beta-Lactamase inhibitory protein (BLIP) is a potent inhibitor of several beta-lactamases including TEM-1 beta-lactamase (Ki = 0.1 nM). The co-crystal structure of TEM-1 beta-lactamase and BLIP has been solved, revealing the contact residues involved in the interface between the enzyme and inhibitor. To determine which residues in TEM-1 beta-lactamase are critical for binding BLIP, the method of monovalent phage display was employed. Random mutants of TEM-1 beta-lactamase in the 99-114 loop-helix and 235-240 B3 beta-strand regions were displayed as fusion proteins on the surface of the M13 bacteriophage. Functional mutants were selected based on the ability to bind BLIP. After three rounds of enrichment, the sequences of a collection of functional beta-lactamase mutants revealed a consensus sequence for the binding of BLIP. Seven loop-helix residues including Asp-101, Leu-102, Val-103, Ser-106, Pro-107, Thr-109, and His-112 and three B3 beta-strand residues including Ser-235, Gly-236, and Gly-238 were found to be critical for tight binding of BLIP. In addition, the selected beta-lactamase mutants A113L/T114R and E240K were found to increase binding of BLIP by over 6- and 11-fold, respectively. Combining these substitutions resulted in 550-fold tighter binding between the enzyme and BLIP with a Ki of 0.40 pM. These results reveal that the binding between TEM-1 beta-lactamase and BLIP can be improved and that there are a large number of sequences consistent with tight binding between BLIP and beta-lactamase.     

Histidine ligands in bacterial metallothionein enhance cluster stability

The cyanobacterial metallothionein (MT) SmtA is the prototype for bacterial MTs and protects against elevated levels of zinc. In contrast to mammalian MTs, bacterial MTs coordinate to metal ions not only via cysteine sulfurs, but unusually for MTs, also via histidine nitrogens. To investigate whether histidine coordination in these metal-sulfur clusters provides advantages over S-coordination only, we mutated the two metal-binding histidine residues in the cyanobacterial MT SmtA from Synechococcus PCC7942 to cysteines. We show that the mutant proteins are still capable of binding up to four zinc ions as is the wild-type protein. However, the mutations perturb protein folding and metal-binding dynamics. Interestingly, several homologues of SmtA also show variations in these two residues. We conclude that histidine residues in Synechococcus PCC7942 SmtA have a stabilising effect due to electrostatic interactions that impact on protein folding and metal cluster charge, and are involved in fine-tuning the reactivity of the bound metal ions.     

Modulation of the zinc(II) center in protein farnesyltransferase by mutagenesis of the zinc(II) ligands

Protein farnesyltransferase (PFTase) is a zinc-containing metalloenzyme that catalyzes the alkylation of cysteine (C) in protein substrates containing a C-terminal "CaaX" motif by farnesyl diphosphate (FPP). In yeast PFTase Zn(II) is coordinated to D307, C309, and H363 in the beta-subunit. The inner coordination sphere of the metal also contains a water molecule to give a net charge of 0 for the tetracoordinate Zn(II) center. When the protein substrate binds, the water molecule is replaced by the thiol of the cysteine residue, and the thiol is deprotonated to generate a Zn(II)-stabilized thiolate in the PFTase.FPP.protein ternary complex for the ensuing prenyl transfer reaction. An expression system was constructed for yeast PFTase containing a His(6) tag at the C-terminus of the beta-subunit to facilitate purification of the wild-type enzyme and site-directed mutants. The amino acids that coordinate Zn(II) were substituted to give a series of mutant PFTases with net charges of +1, 0, -1, and -2 at the Zn(II) center of the ternary enzyme.substrate complexes. Wild-type PFTase and the site-directed mutants were purified as alpha,beta-heterodimers, and each was found to contain an equivalent of Zn(II). All of the mutants were less reactive than wt PFTase (net charge of -1), with the greatest losses of activity seen for the mutants with net charges of 0 and +1. Equilibrium binding experiments with dGCVIA peptide and an unreactive analogue of FPP, (E,E)-2-[2-oxo-2-[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]amino]ethyl]phosphonate (FNP), established that all of the mutants bound an equivalent of the peptide substrate. Like wt PFTase, the pH dependence of K(D) for the mutants did not change significantly between pH 5 and pH 9, indicating that pK(A)s for the thiol moiety in the (mutant PFTase).FNP.peptide complexes were <5. dGSVIA and dG(beta-NH2-Ala)VIA, where the sulfhydryl moiety was replaced by hydroxyl and amino groups, respectively, were not substrates. These experiments suggest a direct relationship between the net charge of the Zn(II) center in PFTase and the reactivity of the peptide thiolate that is alkylated by FPP.     

Molecular interactions of zinc(II) cyclams with polycarboxylato ligands

Four new zinc(II) cyclams of the composition {Zn(L)(tp²⁻) · H₂O}_n (1), {Zn(L)(H₂bta²⁻) · 2H₂O}_n (2), [Zn₂(L)₂(ox²⁻)] 2ClO₄ · 2DMF (3), and Zn(L)(H₂btc⁻)₂ · 2DMF (4), where L = cyclam, tp²⁻ = 1,4-benzenedicarboxylate ion, H₂bta²⁻ = 1,2,4,5-benzenetetracarboxylate ion, ox²⁻ = oxalate ion, DMF = N,N-dimethylformamide, and H₂btc⁻ = 1,3,5-benzenetricarboxylate ion, have been synthesized and structurally characterized by a combination of analytical, spectroscopic and crystallographic methods. The carboxylato ligands in the complexes 1-4 show strong coordination tendencies toward zinc(II) cyclams with hydrogen bonding interactions between the pre-organized N-H groups of the macrocycle and oxygen atoms of the carboxylato ligands. The macrocycles in 1, 2, and 4 adopt trans-III configurations with the appropriate R,R,S,S arrangement of the four chiral nitrogen centers, respectively. However, the complex 3 shows an unusual cis V conformation with the R,R,R,R nitrogen configuration. The finding of strong interactions between the carboxylato ligands and the zinc(II) ions may provide additional knowledge for the improved design of receptor-targeted zinc(II) cyclams in anti-HIV agents.     

Cysteinyl peptide inhibitors of Bacillus cereus zinc beta-lactamase

Several cysteinyl peptides have been synthesised and shown to be reversible competitive inhibitors of the Bacillus cereus metallo-beta-lactamase. The pH dependence of pKi indicates that the thiol anion displaces hydroxide ion from the active site zinc(II). D,D-Peptides bind to the enzyme better than other diastereoisomers, which is compatible with the predicted stereochemistry of the active site.     

Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans

Zinc is an essential trace element involved in a wide range of biological processes and human diseases. Zinc excess is deleterious, and animals require mechanisms to protect against zinc toxicity. To identify genes that modulate zinc tolerance, we performed a forward genetic screen for Caenorhabditis elegans mutants that were resistant to zinc toxicity. Here we demonstrate that mutations of the C. elegans histidine ammonia lyase (haly-1) gene promote zinc tolerance. C. elegans haly-1 encodes a protein that is homologous to vertebrate HAL, an enzyme that converts histidine to urocanic acid. haly-1 mutant animals displayed elevated levels of histidine, indicating that C. elegans HALY-1 protein is an enzyme involved in histidine catabolism. These results suggest the model that elevated histidine chelates zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we demonstrated that dietary histidine promotes zinc tolerance. Nickel is another metal that binds histidine with high affinity. We demonstrated that haly-1 mutant animals are resistant to nickel toxicity and dietary histidine promotes nickel tolerance in wild-type animals. These studies identify a novel role for haly-1 and histidine in zinc metabolism and may be relevant for other animals.     

Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae. II. Catalytic mechanism.

Yeast histidine tRNA guanylyltransferase (TGT) catalyzes in the presence of ATP the addition of GTP to the 5' end of eukaryotic cytoplasmic tRNAHis species. A study of the enzyme mechanism with purified protein showed that during the first step ATP is cleaved to AMP and PPi creating adenylylated TGT. In a second step the activated enzyme forms a stable complex with its cognate tRNA substrate. The 5'-phosphate of the tRNA is adenylylated by nucleotide transfer from the adenylylated guanylyltransferase to form A(5')pp(5')N at the 5'-end of the tRNA. Finally, the 3'-hydroxyl of GTP adds to the activated 5' terminus of the tRNA with the release of AMP. This mechanism of tRNAHis guanylyltransferase is very similar to that of RNA ligases. dATP can substitute for ATP in this reaction. Since among several guanosine compounds active in this reaction GTP is most efficiently added we believe that it is the natural substrate of TGT.     

Cryoenzymology of Bacillus cereus beta-lactamase II

The effects of cryosolvents and subzero temperatures on the metalloenzyme beta-lactamase II from Bacillus cereus have been investigated. Preliminary experiments led to the selection of suitable systems for the study of beta-lactamase II catalysis at low temperatures, namely, cobalt(II) beta-lactamase II hydrolysis of benzylpenicillin in 60% (v/v) ethylene glycol and zinc beta-lactamase II hydrolysis of the chromophoric cephalosporin nitrocefin in 60% (v/v) methanol. Progress curves for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II in 60% (v/v) ethylene glycol at temperatures below -30 degrees C consisted of a transient followed by a steady-state phase. The amplitude of the transient implied a burst whose magnitude was greater than the concentration of enzyme, and the proposed mechanism comprises a branched pathway. The kinetics for the simplest variants of such pathways have been worked out, and the rate constants (and activation parameters) for the individual steps have been determined. The spectrum of the enzyme changed during turnover: when benzylpenicillin was added to cobalt beta-lactamase II, there was a large increase in the cysteine-cobalt(II) charge-transfer absorbance at 333 nm. This increase occurred within the time of mixing, even at -50 degrees C. The subsequent decrease in A333 was characterized by a rate constant that had the same value as the "branching" rate constant of the branched-pathway mechanism. This step is believed to be a change in conformation of the enzyme-substrate complex. Single-turnover experiments utilized the change in A333, and the results were consistent with pre-steady-state and steady-state experiments. When a single-turnover experiment at -48 degrees C was quenched with acid, the low molecular weight component of the intermediate was shown to be substrate. The mechanism advanced for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II involves two noncovalent enzyme-substrate complexes that have been characterized by their electronic absorption spectra. When manganese beta-lactamase II was used, the same features (implying a branched pathway) were evident; these experiments were carried out at ordinary temperatures and did not utilize a cryosolvent. The hydrolysis of nitrocefin by zinc beta-lactamase II has been studied concurrently in 60% (v/v) methanol. Progress curves were triphasic. There were two transients preceding the linear steady-state phase. The stoichiometry of the burst again implied a branched pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histidine residues at the N- and C-termini of alpha-helices: perturbed pKas and protein stability.

A single histidine residue has been placed at either the N-terminus or the C-terminus of each of the two alpha-helices of barnase. The pKa of that histidine residue in each of the four mutants has been determined by 1H NMR. The pKas of the two residues at the C-terminus are, on average, 0.5 unit higher, and those of the residues at the N-terminus are 0.8 unit lower, than the pKa of histidines in unfolded barnase at low ionic strength. The conformational stability of the mutant proteins at different values of pH has been measured by urea denaturation. C-Terminal histidine mutants are approximately 0.6 kcal mol-1 more stable when the introduced histidine is protonated, both at low and high ionic strength. N-Terminal mutants with a protonated histidine residue are approximately 1.1 kcal mol-1 less stable at low ionic strength and 0.5 kcal mol-1 less stable at high ionic strength (1 M NaCl). The low-field 1H NMR spectra of the mutant proteins at low pH suggest that the C-terminal histidines form hydrogen bonds with the protein while the N-terminal histidines do not form the same. The perturbations of pKa and stability result from a combination of different electrostatic environments and hydrogen-bonding patterns at either ends of helices. The value of 0.6 kcal mol-1 represents a lower limit to the favorable electrostatic interaction between the alpha-helix dipole and a protonated histidine residue at the C-terminal end of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of zinc ligands in human seminal plasma

Zinc is an essential trace element for male fertility, especially in the capacitation process of spermatozoa. In the present work, after taking into account the contamination and absorption phenomena that occur during chromatography, we found four different zinc ligands in seminal plasma. Citrate is the main (79%) one, then an unknown low-molecular-weight ligand (15%), albumin (4%), and transferrin (2%). Semen samples were successively fractionated on immobilized Cibacron blue, Sephadex G 75 and G 15, and immobilized concanavalin A.     

Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold

Recently, the zinc metallo-hydrolase family of the beta-lactamase fold has grown quite rapidly, accompanied by the accumulation of sequence and structure data. The variety of the biological functions of the family is higher than expected. In addition, the members often have mosaic structures with additional domains. The family includes class B beta-lactamase, glyoxalase II, arylsulfatase, flavoprotein, cyclase/dehydrase, an mRNA 3'-processing protein, a DNA cross-link repair enzyme, a DNA uptake-related protein, an alkylphosphonate uptake-related protein, CMP-N-acetylneuraminate hydroxylase, the romA gene product, alkylsulfatase, and insecticide hydrolases. In this minireview, the functional and structural varieties of the growing protein family are described.     

Histidine residues at the active site of maize δ-aminolevulinic acid dehydratase

Modification of maize δ-aminolevulinic acid dehydratase (ALAD) by diethylpyrocarbonate (DEP) caused rapid and complete inactivation of the enzyme. The inactivation showed saturation kinetics with a half inactivation time at saturating DEP equal to 0.3 min and K_DEP  0.3 mM. Substrate δ-aminolevulinic acid (ALA) and competitive inhibitor levulinic acid protected against inactivation, thereby indicating that DEP modifies the active site. The modified enzyme showed an increase in absorbance at 240 nm which was lost upon treatment with 0.8 M hydroxylamine. Most of the activity lost by DEP treatment could be restored after treatment with 0.8 M hydroxylamine. The results suggest that DEP modifies 7.4 residues/mole of the enzyme. These histidine residues are essential for catalysis by ALAD.