Leucine aminopeptidase (bovine lens). The relative binding of cobalt and zinc to leucine aminopeptidase and the effect of cobalt substitution on specific activity.

Prolonged incubation of zinc-zinc leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with 0.05 M CoCl2 and M KCl in 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees yields an active enzyme in which 2 g atoms of Co2+ per 54,000 dalton subunit have replaced the Zn2+. Incubation of cobalt-cobalt leucine aminopeptidase with various AnCl2 concentrations or zinc-zinc leucine aminopeptidase with various CoCl2 concentrations in M KCl and 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees demonstrates that Co2+ and Zn2+ compete reversibly for two independent binding sites per subunit for which the ratio of the association constants for Zn2+ and Co2+ (1KZn:1KCo = 1KZn/Co; 2KZn:2KCo = 2KZn/Co) are 115 and 15.9 for sites 1 and 2, respectively. The specific activities of the various species of enzyme with 2 mM L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl and 0.01 M NaHCO3 at pH 7.5 are estimated to be (in micromoles per min per mg) 0.043 for the zinc-zinc. 0.039 for the zinc-cobalt, 0.541 for the cobalt-zinc, and 0.536 for the cobalt-cobalt forms, which implies that activity is affected only when cobalt is substituted at site 1, the "activation site." The site, at which cobalt substitution has no effect on activity, is designated the "structural site." The value of Km for cobalt-cobalt leucine aminopeptidase with L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl at pH 7.5 containing 0.01 M NaHCO3 at 30 degrees is 0.52 mM while Vmax is 0.90 mumol per min per mg. In the additional presence of 1 M KCl, Km is 0.19 mM while Vmax is 0.68 mumol per min per mg.     

Cooperative binding of Mn2+ and non-cooperative binding of Mg2+ to beta-galactosidase (E. coli).

Binding and activity studies with β-galactosidase at various concentrations of free Mn²⁺ and Mg²⁺ indicate that Mn²⁺ binds and activates β-galactosidase in a highly cooperative manner while Mg²⁺ binds and activates non-cooperatively. When the data are plotted by the Hill method, slopes of 3.4 for Mn²⁺ and of 1.0 for Mg²⁺ are obtained. The rate of lactose utilization when Mg²⁺ is bound is more than twice that when Mn²⁺ is bound.     

Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase).

Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.     

Leucine aminopeptidase (bovine lens): Synthesis and kinetic properties of ortho-, meta-, and para-substituted leucyl-anilides

The synthesis of a number of leucyl derivatives of substituted anilides and their properties as substrates and inhibitors of Zn²⁺-Mg²⁺ leucine aminopeptidase (EC 3.4.11.1) at pH 8.5 and 30 °C are described. The compounds include leucyl-X where X is o-, m-, or p-aminobenzenesulfonic acid, o-, m-, or p-anisidine, and m- or p-aminobenzenesulfonyl fluoride. The latter two sulfonyl fluorides, designed to be active site-directed irreversible inhibitors, turned out to be good substrates for leucine aminopeptidase. The K_m and V values of the above compounds as substrates for leucine aminopeptidase are reported. N-Leucyl-m-aminobenzenesulfonate exhibits desirable properties (solubility much greater than K_m, Δ? at 295 nm of 2000 m^?1 cm^?1, and V of 300 μmol min^?1 mg^?1) as a substrate for a spectrophotometric assay of leucine aminopeptidase. With the exception of N-leucyl-p-aminobenzenesulfonate, all of the above compounds are inhibitors of the hydrolysis of leucyl-p-nitroanilide by leucine aminopeptidase with K_i values approximately their K_m values when they are used as substrates. Despite wide variability in steric bulk, chemical composition, and electrical charge of the substituted anilides, the K_m values of the above compounds vary over a narrow range (0.5 to 4.8 mm), which indicates that the leucyl moiety plays the predominant role in the determination of K_m values. Although the K_m values of m- substituents are similar to those of o- substituents, the V values for m-substituents are much greater than those for o- substituents, which suggests that o-substituents interfere with the catalytic process. N-Leucyl-p-aminobenzenesulfonate and N-alanyl-p-aminobenzenesulfonate as well as the nonsubstrate p-aminobenzenesulfonate stimulate rather than inhibit the proteolysis of leucyl-p-nitroanilide. The stimulation has no effect on V but lowers the K_m for the hydrolysis of leucyl-p-nitroanilide, which is compatible with these compounds' serving as nonessential activators.     

Mechanism of action of Mg2+ and Zn2+ on rat placental alkaline phosphatase. I. Studies on the soluble Zn2+ and Mg2+ alkaline phosphatases.

Rat placental alkaline phosphatase (EC 3.1.3.1), a dimer of 135,000 daltons, is strongly activated by Mg2+. However, Zn2+ has to be present on the apoenzyme to obtain this activation. Mg2+ alone is unable to reconstitute functional active sites. Excess Zn2+ which competes for the Mg2+ site leads to a phosphatase with little catalytic activity at alkaline pH but with normal active sites at acidic pH as shown by covalent incorporation of ortho-[32P]phosphate. Two enzyme species with identical functional active sites have been reconstituted that only differ by the presence of Zn2+ or Mg2+ at the effector site. A mechanism is presented by which alkaline phosphatase activity of rat placenta would be controlled by a molecular process involving the interaction of Mg2+ and Zn2+ with the dimeric enzyme molecule.     

Effect of Mg2+ on guanine nucleotide sensitivity of ligand binding to serotonin1A receptors from bovine hippocampus

The serotonin1A (5-HT1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein coupled receptors (GPCRs). We report here that guanine nucleotide sensitivity of agonist binding to hippocampal 5-HT1A receptors is dependent on the concentration of Mg2+. Our results show that agonist binding to 5-HT1A receptors is relatively insensitive to guanine nucleotides in the absence of Mg2+. In contrast to this, the specific antagonist binding is insensitive to guanine nucleotides, even in the presence of Mg2+. These results point out the requirement of an optimal concentration of Mg2+ which could be used in assays toward determining guanine nucleotide sensitivity of ligand binding to GPCRs such as the 5-HT1A receptor. Our results provide novel insight into the requirement and concentration dependence of Mg2+ in relation to guanine nucleotide sensitivity for the 5-HT1A receptor in particular, and GPCRs in general.     

Influence of pH on the Mn2+ activation of and binding to yeast enolase: a functional study.

The influence of pH on the activation of yeast enolase by Mn2+ was measured by steady-state kinetics. The pH influence on the binding of Mn2+ to apoenolase and the enolase-substrate complex was measured by EPR spectroscopy. At pH values above 6.6, activation by Mn2+ is fit by Michaelis-Menten kinetics, but at higher concentrations of Mn2+, inhibition is observed. Under conditions analogous to the kinetic studies, the enzyme binds two Mn2+ per dimer with a Kd in the micromolar range. In the presence of the substrate 2-phosphoglycerate, three thermodynamically distinct cation binding sites per monomer are detected and the binding constants are determined by a fit to the data. As the pH decreases, the reaction velocity decreases and the cation inhibition becomes minimal. Under these conditions, only two Mn2+ binding sites per monomer are observed; the third site must be the inhibitory site. The velocity and kinetic constants are minimally affected by buffer except at pH 5.8 with PIPES. Under these conditions, the velocity is only about 40% that observed with other buffers and only a single binding site for Mn2+ per monomer is detected in the presence or absence of substrate. A direct role in the catalytic mechanism by the second cation is called to question. The binding constant for Mn2+ at site I is independent of pH over the range from 7.5 to 5.2, and the binding at site II increases only slightly over this same pH range. These results indicate that the cation sites at positions I and II contain ligands that are pH independent over this range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pleiotropic effects of ATP.Mg2+ binding in the catalytic cycle of ubiquitin-activating enzyme

Conjugation of ubiquitin and other Class 1 ubiquitin-like polypeptides to specific protein targets serves diverse regulatory functions in eukaryotes. The obligatory first step of conjugation requires ATP-coupled activation of the ubiquitin-like protein by members of a superfamily of evolutionarily related enzymes. Kinetic and equilibrium studies of the human ubiquitin-activating enzyme (HsUba1a) reveal that mutations within the ATP.Mg(2+) binding site have remarkably pleiotropic effects on the catalytic phenotype of the enzyme. Mutation of Asp(576) or Lys(528) results in dramatically impaired binding affinities for ATP.Mg(2+), a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations at neither position affect the affinity of HsUbc2b binding; however, differences in k(cat) values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation. These results confirm that Asp(576) and Lys(528) are important for ATP.Mg(2+) binding but are essential catalytic groups for ubiquitin adenylate transition state stabilization. The latter mechanistic effect explicates the observed loss-of-function phenotype associated with mutation of residues paralogous to Asp(576) within the activating enzymes for other ubiquitin-like proteins.     

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.     

Inhibition kinetics of brain butyrylcholinesterase by Cd2+ and Zn2+, Ca2+ or Mg2+ reactivates the inhibited enzyme

• 1.1. The inhibition kinetics of sheep brain butyrylcholinesterase (BChE) (acylcholine acylhydrolase, EC 3.1.1.8) by Cd²⁺ and Zn²⁺ has been studied.
• 2.2. K_s has been determined as 0.14mM. Cd²⁺ and Zn²⁺ were the hyperbolic mixed-type inhibitors of BChE. Ca²⁺ and Mg²⁺ had no effect on the enzyme activity in the experimental conditions.
• 3.3. But when the enzyme was inhibited by 0.1 mM Cd²⁺ or Zn²⁺, Ca²⁺ and Mg²⁺ reactivated the inhibited form of BChE.

     

NMR analysis of bovine tRNATrp: conformation dependence of Mg2+ binding

NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).     

Effects of Li+ transport and intracellular binding on Li+/Mg2+ competition in bovine chromaffin cells

Li(+) transport, intracellular immobilisation and Li(+)/Mg(2+) competition were studied in Li(+)-loaded bovine chromaffin cells. Li(+) influx rate constants, k(i), obtained by atomic absorption (AA) spectrophotometry, in control (without and with ouabain) and depolarising (without and with nitrendipine) conditions, showed that L-type voltage-sensitive Ca(2+) channels have an important role in Li(+) uptake under depolarising conditions. The Li(+) influx apparent rate constant, k(iapp), determined under control conditions by (7)Li NMR spectroscopy with the cells immobilised and perfused, was much lower than the AA-determined value for the cells in suspension. Loading of cell suspensions with 15 mmol l(-1) LiCl led, within 90 min, to a AA-measured total intracellular Li(+) concentration, [Li(+)](iT)=11.39+/-0.56 mmol (l cells)(-1), very close to the steady state value. The intracellular Li(+) T(1)/T(2) ratio of (7)Li NMR relaxation times of the Li(+)-loaded cells reflected a high degree of Li(+) immobilisation in bovine chromaffin cells, similar to neuroblastoma, but larger than for lymphoblastoma and erythrocyte cells. A 52% increase in the intracellular free Mg(2+) concentration, Delta[Mg(2+)](f)=0.27+/-0.05 mmol (l cells)(-1) was measured for chromaffin cells loaded with the Mg(2+)-specific fluorescent probe furaptra, after 90-min loading with 15 mmol l(-1) LiCl, using fluorescence spectroscopy, indicating significant displacement of Mg(2+) by Li(+) from its intracellular binding sites. Comparison with other cell types showed that the extent of intracellular Li(+)/Mg(2+) competition at the same Li(+) loading level depends on intracellular Li(+) transport and immobilisation in a cell-specific manner, being maximal for neuroblastoma cells.     

Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport of red blood cell membranes

Red blood cells contain a protein that activates membrane-bound (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175–4184, 1977] described a modulator-binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator-binding protein was shown to antagonize the activation of (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.     

Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease.

The EcoRV mutant D90A which carries an amino acid substitution in its active center does not cleave DNA. Therefore, it is possible to perform DNA binding experiments with the EcoRV-D90A mutant both in the absence and in the presence of Mg2+. Like wild-type EcoRV [Taylor et al. (1991) Biochemistry 30, 8743-8753], it does not show a pronounced specificity for binding to its recognition site in the absence of Mg2+ as judged by the appearance of multiple shifted bands in an electrophoretic mobility shift assay with a 377-bp DNA fragment carrying a single EcoRV recognition sequence. In the presence of Mg2+, however, only one band corresponding to a 1:1 complex appears even with a high excess of protein over DNA. This complex most likely is the specific one, because its formation is suppressed much more effectively by a 13-bp oligodeoxynucleotide with an EcoRV site than by a corresponding oligodeoxynucleotide without an EcoRV site. The preferential interaction of the EcoRV-D90A mutant with specific DNA in the presence of Mg2+ was also demonstrated directly: a 20-bp oligodeoxynucleotide with an EcoRV site is bound with KAss = 4 x 10(8) M-1, while a corresponding oligodeoxynucleotide without an EcoRV site is bound with KAss less than or equal to 1 x 10(5) M-1. From these data it appears that Mg2+ confers DNA binding specificity to this mutant by lowering the affinity to nonspecific sites and raising the affinity to specific sites as compared to binding in the absence of Mg2+. It is concluded that this is also true for wild-type EcoRV.