Leptin modulates pancreatic β-cell membrane potential through Src kinase–mediated phosphorylation of NMDA receptors

The adipocyte-derived hormone leptin increases trafficking of K_ATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca²⁺ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.     

The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of approximately 200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis.     

Location of the redox-active cysteines in chloroplast sedoheptulose-1,7-bisphosphatase indicates that its allosteric regulation is similar but not identical to that of fructose-1,6-bisphosphatase

Sedoheptulose-1,7-bisphosphatase (SBPase) is a Calvin Cycle enzyme exclusive to chloroplasts and is involved in photosynthetic carbon fixation. The two cysteine residues involved in its redox regulation have been identified by site-directed mutagenesis. They are four residues apart in a predicted loop between two alpha helices and probably form a disulphide bond when oxidised. Three-dimensional modelling of SBPase has been performed using crystallographic data from the structurally homologous pig fructose-1,6-bisphosphatase (FBPase). The results suggest that formation of the disulphide bridge in SBPase is directly analogous to the allosteric regulation of pig FBPase by AMP in terms of the resulting structural changes. Similar changes are thought to occur in chloroplast FBPase, which like SBPase, is also redox regulated and involved in carbon fixation. From the results presented here it appears that the same basic mechanism for the allosteric regulation of enzymic activity operates in the FBPases and SBPase but that the sites at which the regulatory ligands (AMP or thioredoxin) exert their effects are different in each     

Kinetics of the oxidation of ascorbic acid, ferrocyanide and p-phenolsulfonic acid by chloroperoxidase compounds I and II

For the first time elementary reactions involving chloroperoxidase compounds I and II have been investigated. A multi-mixing stopped-flow apparatus was used to study the kinetics of the reactions of compounds I and II with ascorbic acid, ferrocyanide and p-phenolsulfonic acid. The second-order rate constants of the reactions of both compounds with all three substrates were determined between pH 3 and pH 7. In all cases the rate constants decrease with increasing pH. The reactions of p-phenolsulfonic acid are influenced by a catalytically important group on both compounds I and II with a pKa of 3.7 +/- 0.2. With ascorbic acid and ferrocyanide as substrates, a decrease in rate was observed upon ionization of the substrate. Comparisons with horseradish peroxidase show that chloroperoxidase is a much less efficient peroxidatic enzyme. The kinetic data were used to calculate the percentage composition of the mixture of chloroperoxidase species which contribute to the spectra measured during the turnover with ascorbate as substrate.     

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 degrees C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I-NADH reaction can be explained in terms of a single ionization of pKa = 4.7 +/- 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-II-NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 +/- 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II-NADH reaction was observed. Over the pH range of 4-10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 X 10(5) to 5.6 X 10(2) M-1 s-1 and of HRP-II with NADH varied from 4.4 X 10(4) to 4.1 M-1 s-1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.     

Cytochrome c peroxidase activity of a protease-modified form of cytochrome c-552 from the denitrifying bacterium Pseudomonas perfectomarina

Protease activity present in aerobically grown cells of Pseudomonas perfectomarina, protease apparently copurified with cytochrome c-552, and trypsin achieved a limited proteolysis of the diheme cytochrome c-552. That partial lysis conferred cytochrome c peroxidase activity upon cytochrome c-552. The removal of a 4000-Da peptide explains the structural changes in the cytochrome c-552 molecule that resulted in the appearance of both cytochrome c peroxidase activity (with optimum activity at pH 8.6) and a high-spin heme iron. The oxidized form of the modified cytochrome c-552 bound cyanide to the high-spin ferric heme with a rate constant of (2.1 +/- 0.1) X 10(3) M-1 s-1. The dissociation constant was 11.2 microM. Whereas the intact cytochrome c-552 molecule can be half-reduced by ascorbate, the cytochrome c peroxidase was not reducible by ascorbate, NADH, ferrocyanide, or reduced azurin. Dithionite reduced the intact protein completely but only half-reduced the modified form. The apparent second-order rate constant for dithionite reduction was (7.1 +/- 0.1) X 10(2) M-1 s-1 for the intact protein and (2.2 +/- 0.1) X 10(3) M-1 s-1 for the modified form. In contrast with other diheme cytochrome c peroxidases, reduction of the low-spin heme was not necessary to permit ligand binding by the high-spin heme iron.     

Mechanism of horseradish peroxidase-catalyzed oxidation of malonaldehyde

The mechanism of malonaldehyde oxidation by horseradish peroxidase in the presence of manganese(II) and acetate was investigated. Our results show that an apparent oxygenase behavior demonstrated by peroxidase in this system can be explained in terms of normal peroxidase activity. A free radical is generated from the reaction of malonaldehyde with compounds I and II of peroxidase; this radical is scavenged by dissolved molecular oxygen to give the appearance of peroxidase acting as an oxygenase. Oxygen consumption, absorbance spectra, and kinetic results show that the reaction is initiated by autoxidation of malonaldehyde to give a free radical. The radical reacts with oxygen and through the action of manganese(II), a peroxide is generated. This peroxide drives the peroxidase cycle to generate more free radicals which propagate the oxygen consumption reaction.     

Evidence for a peroxidatic oxidation of norepinephrine, a catecholamine, by lactoperoxidase

The electron spin resonance-spin stabilization technique has been applied to identify the o-semiquinone intermediate produced during the lactoperoxidase-catalyzed oxidation of the catecholamine norepinephrine. The results of a rapid scan and spectrophotometric investigation of the reaction clearly indicate a normal peroxidatic pathway of catecholamine degradation.     

Cyanide binding to canine myeloperoxidase

Equilibria and kinetics of cyanide binding to canine myeloperoxidase were studied. Spectral results support the presence of two heme binding sites; an isosbestic point at 444 nm and a linear Scatchard plot suggest that the binding affinity of cyanide to the two subunits of the enzyme is the same. The dissociation constant is 0.53 microM. The pH dependence of the apparent second order rate constant indicates the presence of an acid-base group on the enzyme with a pKa of 3.8 +/- 0.1. The protonated form of cyanide binds to the basic enzyme with a rate constant of (4.3 +/- 0.3) x 10(6) M-1 s-1.     

Mechanism of azide binding to chloroperoxidase and horseradish peroxidase: use of an iodine laser temperature-jump apparatus

The kinetics of azide binding to chloroperoxidase have been studied at eight pH values ranging from 3.0 to 6.6 at 9.5 +/- 0.2 degrees C and ionic strength of 0.4 M in H2O. The same reaction was studied in D2O at pD 4.36. In addition, results were obtained on azide binding to horseradish peroxidase at pD 4.36 and pH 4.56. Typical relaxation times were in the range 10-40 microseconds. The value of kH/kD(on) for chloroperoxidase is 1.16, and kH/kD(off) is 1.7; corresponding values for horseradish peroxidase are 1.10 and 2.4. The H/D solvent isotope effects indicate proton transfer is partially rate controlling and is more important in the dissociation of azide from the enzyme-ligand complex. A mechanism is proposed in which hydrazoic acid binds to chloroperoxidase in a concerted process in which its proton is transferred to a distal basic group. Hydrogen bonding from the newly formed distal acid to the bound azide facilitates formation of hydrazoic acid as the leaving group in the dissociation process. The binding rate constant data, kon, can be fit to the equation kon = k3/(1 + KA/[H+]), where k3 = 7.6 X 10(7) M-1 S-1 and KA, the dissociation constant of hydrazoic acid, is 2.5 X 10(-5) M. The same mechanism probably is valid for the ligand binding to horseradish peroxidase.