Gu-Gang Chang

Selective Oxidative Modification and Affinity Cleavage of Pigeon Liver Malic Enzyme by the Cu2+-Ascorbate System

Pigeon liver malic enzyme was rapidly inactivated by micromolar concentration of Fe2+ in the presence of ascorbate at neutral pH. The inactivated enzyme was subsequently cleaved by the Fe2+-ascorbate system at the chemical bond between Asp258 and Ile259 (Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., and Chang, G. G.(1994) Biochemistry, 33, 7931-7936), which was confirmed by site-specific mutagenesis (Wei, C. H., Chou, W. Y., and Chang, G. G.(1995) Biochemistry 34, 7949-7954). In the present study, at neutral pH, Cu2+ was found to be more reactive in the oxidative modification of malic enzyme and the enzyme was cleaved in a similar manner as Fe2+ did. At acidic pH, however, Fe2+ was found to be ineffective in oxidative modification of the enzyme. Nevertheless, Cu2+ still caused enzyme inactivation and cleaved the enzyme at Asp141-Gly142, Asp194-Pro195, or Asp464-Asp465. Mn2+ and L-malate synergistically protect the enzyme from Cu2+ inactivation at acidic pH. Cu2+ is also a competitive inhibitor versus Mn2+ in the malic enzyme-catalyzed reaction with Ki value 70.3 ± 5.8 μM. The above results indicated that, in addition to the previously determined Asp258 at neutral pH, Asp141, Asp194, and Asp464 are also the coordination sites for the metal binding of malic enzyme. We suggest that the mechanism of affinity modification and cleavage of malic enzyme by the Cu2+-ascorbate system proceed in the following sequence. First, Cu2+ binds with the enzyme at the Mn2+ binding site and reduces to Cu+ by ascorbate. Next, the local oxygen molecules are reduced by Cu+, thereby generating superoxide or other reactive free radicals. These radicals interact with the susceptible essential amino acid residues at the metal-binding site, ultimately causing enzyme inactivation. Finally, the modified enzyme is cleaved into several peptide fragments, allowing the identification of metal site of the enzyme. The pH-dependent different specificities of metal-catalyzed oxidation system may be generally applicable for other enzymes or proteins.

Structural Studies of the Pigeon Cytosolic Nadp+ -Dependent Malic Enzyme

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2 in the presence of divalent cations (Mg2+, Mn2+). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD+ or NADP+, but not both. Structural studies of the human mitochondrial NAD+‐dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP+‐dependent malic enzyme, in a closed form, in a quaternary complex with NADP+, Mn2+, and oxalate. This represents the first structural information on an NADP+‐dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2′‐phosphate group of the NADP+ cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP+ selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.

Critical Assessment of Important Regions in the Subunit Association and Catalytic Action of the Severe Acute Respiratory Syndrome Coronavirus Main Protease

The severe acute respiratory syndrome (SARS) coronavirus (CoV) main protease represents an attractive target for the development of novel anti-SARS agents. The tertiary structure of the protease consists of two distinct folds. One is the N-terminal chymotrypsin-like fold that consists of two structural domains and constitutes the catalytic machinery; the other is the C-terminal helical domain, which has an unclear function and is not found in other RNA virus main proteases. To understand the functional roles of the two structural parts of the SARS-CoV main protease, we generated the full-length of this enzyme as well as several terminally truncated forms, different from each other only by the number of amino acid residues at the C- or N-terminal regions. The quaternary structure and Kd value of the protease were analyzed by analytical ultracentrifugation. The results showed that the N-terminal 1–3 amino acid-truncated protease maintains 76% of enzyme activity and that the major form is a dimer, as in the wild type. However, the amino acids 1–4-truncated protease showed the major form to be a monomer and had little enzyme activity. As a result, the fourth amino acid seemed to have a powerful effect on the quaternary structure and activity of this protease. The last C-terminal helically truncated protease also exhibited a greater tendency to form monomer and showed little activity. We concluded that both the C- and the N-terminal regions influence the dimerization and enzyme activity of the SARS-CoV main protease.

Structural and Functional Variations in Human Apolipoprotein E3 and E4

There are three major apolipoprotein E (apoE) isoforms. Although APOE-ϵ3 is considered a longevity gene, APOE-ϵ4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N- and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease.

Determinants of the Dual Cofactor Specificity and Substrate Cooperativity of the Human Mitochondrial NAD(P)+-dependent Malic Enzyme FUNCTIONAL ROLES OF GLUTAMINE 362

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The ΔG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.

Investigation of the Dimer Interface and Substrate Specificity of Prolyl Dipeptidase DPP8

DPP8 belongs to the family of prolyl dipeptidases, which are capable of cleaving the peptide bond after a penultimate proline residue. Unlike DPP-IV, a drug target for type II diabetes, no information is available on the crystal structure of DPP8, the regulation of its enzymatic activity, or its substrate specificity. In this study, using analytical ultracentrifugation and native gel electrophoresis, we show that the DPP8 protein is predominantly dimeric when purified or in the cell extracts. Four conserved residues in the C-terminal loop of DPP8 (Phe822, Val833, Tyr844, and His859), corresponding to those located at the dimer interface of DPP-IV, were individually mutated to Ala. Surprisingly, unlike DPP-IV, these single-site mutations abolished the enzymatic activity of DPP8 without disrupting its quaternary structure, indicating that dimerization itself is not sufficient for the optimal enzymatic activity of DPP8. Moreover, these mutations not only decreased kcat, as did the corresponding DPP-IV mutations, but also dramatically increased Km. We further show that the Km effect is independent of the substrate assayed. Finally, we identified the distinctive and strict substrate selectivity of DPP8 for hydrophobic or basic residues at the P2 site, which is in sharp contrast to the much less discriminative substrate specificity of DPP-IV. Our study has identified the residues absolutely required for the optimal activity of DPP8 and its unique substrate specificity. This study extends the functional importance of the C-terminal loop to the whole family of prolyl dipeptidases.

Differentiation of the slow‐binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase

The binding mechanism of Mg2+ at the M3 site of human placental alkaline phosphatase was found to be a slow‐binding process with a low binding affinity (KMg(app.) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg2+‐free and Mg2+‐containing enzymes by acrylamide showed almost identical dynamic quenching constant (Ksv = 4.44 ± 0.09 M−1), indicating that there is no gross conformational difference between the M3‐free and the M3‐Mg2+ enzymes. However, Zn2+ was found to have a high affinity with the M3 site (KZn(app.) = 0.11 mM) and was observed as a time‐dependent inhibitor of the enzyme. The dependence of the observed transition rate from higher activity to lower activity (kobs) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational change of the enzyme, which locks the enzyme in a conformation (M3′‐Zn) having an extremely high affinity for the Zn2+ (K*Zn(app.) = 0.33 μM). The conformation of the M3′‐Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg2+ activation and Zn2+ inhibition of the enzyme are reversible processes. Structural information indicates that the M3 site, which is octahedrally coordinated to Mg2+, has been converted to a distorted tetrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching constant for acrylamide (Ksv = 3.86 ± 0.04 M−1), suggesting a conformational change. Both Mg2+ and phosphate prevent the enzyme from reaching this inactive structure. GTP plays an important role in reactivating the Zn‐inhibited enzyme activity. We propose that, under physiological conditions, magnesium ion may play an important modulatory role in the cell for protecting the enzyme by retaining a favorable geometry of the active site needed for catalysis.

Kinetic mechanism of the cytosolic malic enzyme from human breast cancer cell line

The kinetic mechanism of the cytosolic NADP(+)-dependent malic enzyme from cultured human breast cancer cell line was studied by steady-state kinetics. In the direction of oxidative decarboxylation, the initial-velocity and product-inhibition studies indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism with NADP+ as the leading substrate followed by L-malate. The products are released in the order of CO2, pyruvate, and NADPH. The enzyme is unstable at high salt concentration and elevated temperature. However, it is stable for at least 20 min under the assay conditions. Tartronate (2-hydroxymalonate) was found to be a noncompetitive inhibitor for the enzyme with respect to L-malate. The kinetic mechanism of the cytosolic tumor malic enzyme is similar to that for the pigeon liver cytosolic malic enzyme but different from those for the mitochondrial enzyme from various sources.

Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)

Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the K(m) values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate K(m) values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The K(act) values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.

Mutation of Glu-166 Blocks the Substrate-Induced Dimerization of SARS Coronavirus Main Protease

Abstract The maturation of SARS coronavirus involves the autocleavage of polyproteins 1a and 1ab by the main protease (Mpro) and a papain-like protease; these represent attractive targets for the development of anti-SARS drugs. The functional unit of Mpro is a homodimer, and each subunit has a His-41⋯Cys-145 catalytic dyad. Current thinking in this area is that Mpro dimerization is essential for catalysis, although the influence of the substrate binding on the dimer formation has never been explored. Here, we delineate the contributions of the peptide substrate to Mpro dimerization. Enzyme kinetic assays indicate that the monomeric mutant R298A/L exhibits lower activity but in a cooperative manner. Analytical ultracentrifugation analyses indicate that in the presence of substrates, the major species of R298A/L shows a significant size shift toward the dimeric form and the monomer-dimer dissociation constant of R298A/L decreases by 12- to 17-fold, approaching that for wild-type. Furthermore, this substrate-induced dimerization was found to be reversible after substrates were removed. Based on the crystal structures, a key residue, Glu-166, which is responsible for recognizing the Gln-P1 of the substrate and binding to Ser-1 of another protomer, will interact with Asn-142 and block the S1 subsite entrance in the monomer. Our studies indicate that mutation of Glu-166 in the R298A mutant indeed blocks the substrate-induced dimerization. This demonstrates that Glu-166 plays a pivotal role in connecting the substrate binding site with the dimer interface. We conclude that protein-ligand and protein-protein interactions are closely correlated in Mpro.