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Biomedical subjects

Li-June Ming

Publications and source records attributed to Li-June Ming.

12 recordsLinked to original sources
PubMed2006

A "moonlighting" dizinc aminopeptidase from Streptomyces griseus: mechanisms for peptide hydrolysis and the 4 x 10(10)-fold acceleration of the alternative phosphodiester hydrolysis.

A unique "enzyme catalytic promiscuity" has recently been observed, wherein a phosphodiester and a phosphonate ester are hydrolyzed by a dinuclear aminopeptidase and its metal derivatives from Streptomyces griseus (SgAP) [Park, H. I., Ming, L.-J. (1999) Angew. Chem., Int. Ed. Engl. 38, 2914-2916 and Ercan, A., Park, H. I., Ming, L.-J. (2000) Chem. Commun. 2501-2502]. Because tetrahedral phosphocenters often serve as transition-state inhibitors toward the hydrolysis of the peptide, phosphoester hydrolysis by peptidases is thus not expected to occur effectively and must take place through a unique mechanism. Owing to the very different structures and mechanistic requirements between phosphoesters and peptides during hydrolysis, the study of this effective phosphodiester hydrolysis by SgAP may provide further insight into the action of this enzyme that is otherwise not obtainable from regular peptide substrates. We present herein a detailed investigation of both peptide and phosphodiester hydrolyses catalyzed by SgAP. The latter exhibits a first-order rate enhancement of 4 x 10(10)-fold compared to the uncatalyzed reaction at pH 7.0 and 25 degrees C. The results suggest that peptide and phosphodiester hydrolyses by SgAP may share a common reaction mechanism to a certain extent. However, their differences in pH dependence, phosphate and fluoride inhibition patterns, and proton inventory reflect that they must follow different pathways. Mechanisms for the two hydrolyses are drawn on the basis of the results, which provide the foundation for further investigation of the catalytic promiscuity of this enzyme by means of physical and molecular biology methods. The catalytic versatility of SgAP suggests that this enzyme may serve as a unique "natural model system" for further investigation of dinuclear hydrolysis. A better understanding of enzyme catalytic promiscuity is also expected to shed light on the evolution and action of enzymes.

Amino Acid Sequence↗
PubMed2006

Overexpression and mechanistic characterization of blastula protease 10, a metalloprotease involved in sea urchin embryogenesis and development.

Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1. Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated "tyrosine switch." It has not been determined if the more structurally complex members of this family involved in eukaryotic development share this mechanism. The recombinant BP10 has been overexpressed in Escherichia coli, its metalloenzyme nature has been confirmed, and its catalytic properties have been characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native zinc-containing form and copper derivative. The copper derivative of BP10 shows a remarkable 960% rate acceleration toward the hydrolysis of the synthetic substrate N-benzoyl-arginine-p-nitroanilide when compared with the zinc form. The enzyme also shows calcium-dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-type enzymes in deuterostomes, which can add supporting data to corroborate the metal-centered mechanism proposed for astacin and the role of the coordinated Tyr. We have demonstrated the first mechanistic study of a tolloid-related metalloenzyme involved in sea urchin embryogenesis.

Animals↗
PubMed2005

Catechol oxidase activity of di-Cu2+-substituted aminopeptidase from Streptomyces griseus.

Streptomyces griseus aminopeptidase exhibits activities toward the hydrolyses of peptides and bis(p-nitrophenyl)phosphate (40 billion fold) and catechol oxidation reported herein with catalytic efficiency (kcat/Km) only about 10 times smaller than that of gypsywort catechol oxidase. The multifunctionality of this enzyme suggests that it is a unique system for further exploration of protein structure and function and a template for design of enzymes of diverse activities.

Aminopeptidases↗
PubMed2005

Proton transfer from exogenous donors in catalysis by human carbonic anhydrase II.

In the site-specific mutant of human carbonic anhydrase in which the proton shuttle His64 is replaced with alanine, H64A HCA II, catalysis can be activated in a saturable manner by the proton donor 4-methylimidazole (4-MI). From 1H NMR relaxivities, we found 4-MI bound as a second-shell ligand of the tetrahedrally coordinated cobalt in Co(II)-substituted H64A HCA II, with 4-MI located about 4.5 A from the metal. Binding constants of 4-MI to H64A HCA II were estimated from: (1) NMR relaxation of the protons of 4-MI by Co(II)-H64A HCA II, (2) the visible absorption spectrum of Co(II)-H64A HCA II in the presence of 4-MI, (3) the inhibition by 4-MI of the catalytic hydration of CO2, and (4) from the catalyzed exchange of 18O between CO2 and water. These experiments along with previously reported crystallographic and catalytic data help identify a range of distances at which proton transfer is efficient in carbonic anhydrase II.

Amino Acid Substitution↗
PubMed2005

Catechol oxidase-like oxidation chemistry of the 1-20 and 1-16 fragments of Alzheimer's disease-related beta-amyloid peptide: their structure-activity correlation and the fate of hydrogen peroxide.

The Cu2+ complexes of the 1-16 and the 1-20 fragments of the Alzheimer's disease-related beta-amyloid peptide (CuAbeta) show significant oxidative activities toward a catechol-like substrate trihydroxylbenzene and plasmid DNA cleavage. The latter reflects possible oxidative stress to biological macromolecules, yielding supporting data to the pathological role of these soluble Abeta fragments. The former exhibits enzyme-like kinetics and is dependent on [H2O2], exhibiting k(cat) of 0.066 s-1 (6000-fold higher than the reaction without CuAbeta) and k(cat)/Km of 37.2 m-1s-1 under saturating [H2O2] of approximately 0.24%. This kinetic profile is consistent with metal-centered redox chemistry for the action of CuAbeta. A mechanism is proposed by the use of the catalytic cycle of dinuclear catechol oxidase as a working model. Trihydroxylbenzene is also oxidized by CuAbeta aerobically without H2O2, affording rate constants of 6.50x10(-3) s-1 and 3.25 m-1s-1. This activity is also consistent with catechol oxidase action in the absence of H2O2, wherein the substrate binds and reduces the Cu2+ center first, followed by O2 binding to afford the mu-eta2:eta2-peroxo intermediate, which oxidizes a second substrate to complete the catalytic cycle. A tetragonally distorted octahedral metal coordination sphere with three coordinated His side chains and some specific H-bonding interactions is concluded from the electronic spectrum of CuAbeta, hyperfine-shifted 1H NMR spectrum of CoAbeta, and molecular mechanics calculations. The results presented here are expected to add further insight into the chemistry of metallo-Abeta, which may assist better understanding of the neuropathology of Alzheimer's disease.

Alzheimer Disease↗
PubMed2003

Structure and function of "metalloantibiotics".

Although most antibiotics do not need metal ions for their biological activities, there are a number of antibiotics that require metal ions to function properly, such as bleomycin (BLM), streptonigrin (SN), and bacitracin. The coordinated metal ions in these antibiotics play an important role in maintaining proper structure and/or function of these antibiotics. Removal of the metal ions from these antibiotics can cause changes in structure and/or function of these antibiotics. Similar to the case of "metalloproteins," these antibiotics are dubbed "metalloantibiotics" which are the title subjects of this review. Metalloantibiotics can interact with several different kinds of biomolecules, including DNA, RNA, proteins, receptors, and lipids, rendering their unique and specific bioactivities. In addition to the microbial-originated metalloantibiotics, many metalloantibiotic derivatives and metal complexes of synthetic ligands also show antibacterial, antiviral, and anti-neoplastic activities which are also briefly discussed to provide a broad sense of the term "metalloantibiotics."

Anti-Bacterial Agents↗
PubMed2002

Paramagnetic cobalt(II) as a probe for kinetic and NMR relaxation studies of phosphate binding and the catalytic mechanism of Streptomyces dinuclear aminopeptidase.

Phosphate was proposed to be a bridging ligand in the structure 1xjo.pdb of Streptomyces dizinc aminopeptidase (sAP), which prompted further studies of phosphate binding to this enzyme. Phosphate inhibits sAP and its Co(2+)-substituted derivatives in a noncompetitive manner from pH 6.0 to 9.0, with strongest inhibition observed at lower pHs (K(i) = 0.6, 8.2, and 9.1 mM for ZnZn-, CoCo-, and CoZn-sAP, respectively, at pH 6.0), which indicates that phosphate does not compete with substrate binding to the dinuclear active site and that monobasic phosphate has a higher binding affinity. The inhibition K(i)-pH profiles for phosphate inhibition of both the native and the Co(2+)-substituted derivatives reveal a similar pK(a) around 7.0, reflecting that phosphate binding is not affected by the metal centers of different Lewis acidities. Modification of ZnZn- and CoCo-sAP with the arginine-specific reagent phenylglyoxal reveals a significant weakening in phosphate and substrate binding by showing approximately a 10-fold increase in the dissociation constant K(i) for phosphate binding and approximately 4-8-fold increase in K(m). The catalysis is also influenced by the modification as reflected by a significant decrease in k(cat) in both cases. Furthermore, phosphate and the transition-state inhibitor 1-aminobutyl phosphonate can protect arginine from the modification, strongly suggesting that Arg202 near the active site is involved in phosphate binding and in stabilizing the transition state. The effect on (31)P NMR relaxation of phosphate caused by the paramagnetic metal center in Co(2+)-substituted derivatives of sAP has been measured, which reveals that only one phosphate is bound to sAP with the Co(2+)-(31)P distance in the range of 4.1-4.3 A. The (1)H NMR relaxation of the bulk water signal in the CoCo-sAP sample remains unchanged in the presence of phosphate, further indicating that phosphate may not bind to the active-site metals to displace any metal-bound water/hydroxide. These results strongly support that the phosphate binding site is Arg202 and that this residue plays an important role in the action of sAP.

Aminopeptidases↗
PubMed2002

Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin.

Bacitracin is a widely used metallopeptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(2+) for its biological activity, and has been reported to bind several other transition metal ions, including Mn(2+), Co(2+), Ni(2+), and Cu(2+). Despite the widespread use of bacitracin since its discovery in the early 1940s, the structure-activity relationship of this drug has not been established and the coordination chemistry of its metal complexes was not fully determined until recently. This antibiotic has been suggested to influence cell functioning through more than one route. Since bacterial resistance against bacitracin is still rare despite several decades of widespread use, this antibiotic can serve as an ideal lead for the design of potent peptidyl antibiotics lacking bacterial resistance. In this review, the results of physical (including NMR, EPR, and EXAFS) and molecular biological studies regarding the synthesis and structure of bacitracin, the coordination chemistry of its metal derivatives, the mechanism of its antibiotic actions, its influence on membrane function, and its structure and function relationship are discussed.

Anti-Bacterial Agents↗
PubMed2002

Mechanistic studies of the astacin-like Serratia metalloendopeptidase serralysin: highly active (>2000%) Co(II) and Cu(II) derivatives for further corroboration of a "metallotriad" mechanism.

Serralysin is a bacterial Zn-endopeptidase which has been considered a virulence factor to cause tissue damage and anaphylactic response. It contains a coordinated Tyr that is unique to the astacin-like Zn enzymes. The coordinated Tyr has been proposed to play an important role in the action of this endopeptidase family. Several metal-substituted derivatives of serralysin (including Mn2+, Co2+, Ni2+, Cu2+, and Cd2+ derivatives) are found to exhibit significant activities. Particularly, the Co- and Cu-substituted derivatives exhibit much higher activities than the native serralysin toward the hydrolysis of the tripeptide mimic benzoyl-Arg- p-nitroanilide, i.e., 35 and 49 times higher in k(cat) and 33 and 26 times in k(cat)/ K(m), respectively. Such remarkably higher activities of metal-substituted derivatives, especially the Cu derivative, than that of the native Zn enzyme are rare in the literature, reflecting the uniqueness of this enzyme among all Zn enzymes. The significantly different k(cat) yet similar K(m) values among the several metal derivatives suggests that the metal center is involved in catalysis, but not necessarily in the binding of the substrate, whereas the dramatically different inhibition constants for Arg-hydroxamate binding to the metal-substituted derivatives indicates direct binding of this inhibitor to the metal center. The activity-pH profiles of serralysin and its Co2+ and Cu2+ derivatives and the optical-pH profile of Cu-serralysin have been obtained, in which the decrease in activity at higher pH values was found to be associated with a dramatic increase in the Tyr-to-Cu2+ charge transfer transitions. This observation suggests that the binding of Tyr216 to the metal is inhibitory. A metal-centered mechanism is proposed for serralysin catalysis based on the results presented here, in which the detachment of the coordinated Tyr and formation of a H-bond with the transition-state complex are considered essential for the stabilization of the transition state.

Arginine↗
PubMed1999

NMR Study of Dendrimer Structures Using Paramagnetic Cobalt(II) as a Probe.

Cobalt(II) has been utilized as an external paramagnetic (1)H NMR probe for the study of the structure of dendrimers that possess specifically located metal recognition sites. The isotropically shifted (1)H NMR signals of the Co(II) complexes of two 2,6-diamidopyridine-containing dendrimers have been fully assigned by means of 1D and 2D NMR techniques, including NOE difference, EXSY, COSY, and TOCSY. T(1) values of the isotropically shifted signals were used to calculate metal-proton distances to build a molecular model of the internal structure of the dendrimers. The presence of sizable cavities within the dendrimers was observed, including a loosely packed conformation for the 2,6-diamidopyridine moiety to bind to potential guest molecules.

Journal Article↗
PubMed1998

Comprehensive 2D (1)H NMR Studies of Paramagnetic Lanthanide(III) Complexes of Anthracycline Antitumor Antibiotics.

The binding of several lanthanide(III) ions to anthracycline antitumor antibiotics daunomycin and adriamycin in methanol and aqueous solutions has been studied by means of optical and 2D NMR (COSY, TOCSY, and EXSY) techniques. These results indicate that a 1:1 Yb(3+)-drug complex (1) is the predominant complex at a metal-to-ligand ratio <10 with slightly higher proton activities, e.g., approximately pH 4-5 in an aqueous solution. In the presence of a base, a 1:2 (2) or 1:3 (3) Yb(3+)-drug complex can be formed. In addition, a 2:1 complex (4) is formed when the metal-to-drug ratio is >25. These Yb(3+)-drug complexes undergo slow chemical exchange with each other relative to the NMR time scale. Therefore, 1D and 2D magnetization transfer experiments can be utilized for the assignment of the isotropically shifted signals arising from the drug nuclei in the various paramagnetic complexes. The spin-lattice (T(1)) relaxation times and solution magnetic susceptibilities of these Yb(3+)-drug complexes confirmed the binding of the metal ion to 11,12-beta-ketophenolate in all the complexes (except the second Yb(3+) in the 2:1 complex which binds to the 5,6-beta-ketophenolate). Several other lanthanide(III) ions Pr(3+), Eu(3+), and Dy(3+) show similar binding properties to daunomycin based on optical and NMR studies. The binding of Yb(3+) to daunomycin has a profound effect on the reduction potential of the drug, showing a decrease in the potential by 150 mV upon addition of 1 equiv of Yb(3+) to the drug solution. This observation indicates that metal ions must play a significant role in the action of these family of drugs in vivo.

Journal Article↗