Glycerol 3-Phosphate Dehydrogenase: Role of the Protein Conformational Change in Activation of a Readily Reversible Enzyme-Catalyzed Hydride Transfer Reaction.

Kinetic parameters are reported for glycerol 3-phosphate dehydrogenase (GPDH)-catalyzed hydride transfer from the whole substrate glycerol 3-phosphate (G3P) or truncated substrate ethylene glycol (EtG) to NAD, and for activation of the hydride transfer reaction of EtG by phosphite dianion. These kinetic parameters were combined with parameters for enzyme-catalyzed hydride transfer in the microscopic reverse direction to give the reaction equilibrium constants Keq. Hydride transfer from G3P is favored in comparison to EtG because the carbonyl product of the former reaction is stabilized by hyperconjugative electron donation from the -CH2R keto substituent. The kinetic data show that the phosphite dianion provides the same 7.6 ± 0.1 kcal/mol stabilization of the transition states for enzyme-catalyzed reactions in the forward [reduction of NAD by EtG] and reverse [oxidation of NADH by glycolaldehyde] directions. The experimental evidence that supports a role for phosphite dianion in stabilizing the active closed form of the GPDH (EC) relative to the ca. 6 kcal/mol more unstable open form (EO) is summarized.

Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution.

Many enzymes that show a large specificity in binding the enzymatic transition state with a higher affinity than the substrate utilize substrate binding energy to drive protein conformational changes to form caged substrate complexes. These protein cages provide strong stabilization of enzymatic transition states. Using part of the substrate binding energy to drive the protein conformational change avoids a similar strong stabilization of the Michaelis complex and irreversible ligand binding. A seminal step in the development of modern enzyme catalysts was the evolution of enzymes that couple substrate binding to a conformational change. These include enzymes that function in glycolysis (triosephosphate isomerase), the biosynthesis of lipids (glycerol phosphate dehydrogenase), the hexose monophosphate shunt (6-phosphogluconate dehydrogenase), and the mevalonate pathway (isopentenyl diphosphate isomerase), catalyze the final step in the biosynthesis of pyrimidine nucleotides (orotidine monophosphate decarboxylase), and regulate the cellular levels of adenine nucleotides (adenylate kinase). The evolution of enzymes that undergo ligand-driven conformational changes to form active protein-substrate cages is proposed to proceed by selection of variants, in which the selected side chain substitutions destabilize a second protein conformer that shows compensating enhanced binding interactions with the substrate. The advantages inherent to enzymes that incorporate a conformational change into the catalytic cycle provide a strong driving force for the evolution of flexible protein folds such as the TIM barrel. The appearance of these folds represented a watershed event in enzyme evolution that enabled the rapid propagation of enzyme activities within enzyme superfamilies.

Glycerol-3-Phosphate Dehydrogenase: The K120 and K204 Side Chains Define an Oxyanion Hole at the Enzyme Active Site.

The cationic K120 and K204 side chains lie close to the C-2 carbonyl group of substrate dihydroxyacetone phosphate (DHAP) at the active site of glycerol-3-phosphate dehydrogenase (GPDH), and the K120 side chain is also positioned to form a hydrogen bond to the C-1 hydroxyl of DHAP. The kinetic parameters for unactivated and phosphite dianion-activated GPDH-catalyzed reduction of glycolaldehyde and acetaldehyde (AcA) show that the transition state for the former reaction is stabilized by ca 5 kcal/mole by interactions of the C-1 hydroxyl group with the protein catalyst. The K120A and K204A substitutions at wild-type GPDH result in similar decreases in kcat, but Km is only affected by the K120A substitution. These results are consistent with 3 kcal/mol stabilizing interactions between the K120 or K204 side chains and a negative charge at the C-2 oxygen at the transition state for hydride transfer from NADH to DHAP. This stabilization resembles that observed at oxyanion holes for other enzymes. There is no detectable rescue of the K204A variant by ethylammonium cation (EtNH3+), compared with the efficient rescue of the K120A variant. This is consistent with a difference in the accessibility of the variant enzyme active sites to exogenous EtNH3+. The K120A/K204A substitutions cause a (6 × 106)-fold increase in the promiscuity of wild-type hlGPDH for catalysis of the reduction of AcA compared to DHAP. This may reflect conservation of the active site for an ancestral alcohol dehydrogenase, whose relative activity for catalysis of reduction of AcA increases with substitutions that reduce the activity for reduction of the specific substrate DHAP.

Hydride Transfer Catalyzed by Glycerol Phosphate Dehydrogenase: Recruitment of an Acidic Amino Acid Side Chain to Rescue a Damaged Enzyme.

K120 of glycerol 3-phosphate dehydrogenase (GPDH) lies close to the carbonyl group of the bound dihydroxyacetone phosphate (DHAP) dianion. pH rate (pH 4.6-9.0) profiles are reported for kcat and (kcat/Km)dianion for wild type and K120A GPDH-catalyzed reduction of DHAP by NADH, and for (kcat/KdKam) for activation of the variant-catalyzed reduction by CH3CH2NH3+, where Kam and Kd are apparent dissociation constants for CH3CH2NH3+ and DHAP, respectively. These profiles provide evidence that the K120 side chain cation, which is stabilized by an ion-pairing interaction with the D260 side chain, remains protonated between pH 4.6 and 9.0. The profiles for wild type and K120A variant GPDH show downward breaks at a similar pH value (7.6) that are attributed to protonation of the K204 side chain, which also lies close to the substrate carbonyl oxygen. The pH profiles for (kcat/Km)dianion and (kcat/KdKam) for the K120A variant show that the monoprotonated form of the variant is activated for catalysis by CH3CH2NH3+ but has no detectable activity, compared to the diprotonated variant, for unactivated reduction of DHAP. The pH profile for kcat shows that the monoprotonated K120A variant is active toward reduction of enzyme-bound DHAP, because of activation by a ligand-driven conformational change. Upward breaks in the pH profiles for kcat and (kcat/Km)dianion for K120A GPDH are attributed to protonation of D260. These breaks are consistent with the functional replacement of K120 by D260, and a plasticity in the catalytic roles of the active site side chains.

The Organization of Active Site Side Chains of Glycerol-3-phosphate Dehydrogenase Promotes Efficient Enzyme Catalysis and Rescue of Variant Enzymes.

A comparison of the values of kcat/Km for reduction of dihydroxyacetone phosphate (DHAP) by NADH catalyzed by wild type and K120A/R269A variant glycerol-3-phosphate dehydrogenase from human liver (hlGPDH) shows that the transition state for enzyme-catalyzed hydride transfer is stabilized by 12.0 kcal/mol by interactions with the cationic K120 and R269 side chains. The transition state for the K120A/R269A variant-catalyzed reduction of DHAP is stabilized by 1.0 and 3.8 kcal/mol for reactions in the presence of 1.0 M EtNH3+ and guanidinium cation (Gua+), respectively, and by 7.5 kcal/mol for reactions in the presence of a mixture of each cation at 1.0 M, so that the transition state stabilization by the ternary E·EtNH3+·Gua+ complex is 2.8 kcal/mol greater than the sum of stabilization by the respective binary complexes. This shows that there is cooperativity between the paired activators in transition state stabilization. The effective molarities (EMs) of ∼50 M determined for the K120A and R269A side chains are ≪106 M, the EM for entropically controlled reactions. The unusually efficient rescue of the activity of hlGPDH-catalyzed reactions by the HPi/Gua+ pair and by the Gua+/EtNH3+ activator pair is due to stabilizing interactions between the protein and the activator pieces that organize the K120 and R269 side chains at the active site. This "preorganization" of side chains promotes effective catalysis by hlGPDH and many other enzymes. The role of the highly conserved network of side chains, which include Q295, R269, N270, N205, T264, K204, D260, and K120, in catalysis is discussed.

The unusual di-domain structure of Dunaliella salina glycerol-3-phosphate dehydrogenase enables direct conversion of dihydroxyacetone phosphate to glycerol.

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di-domain glycerol-3-phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase-like domain fused to the canonical glycerol-3-phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co-factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.

Characterization and diverse evolution patterns of glycerol-3-phosphate dehydrogenase family genes in Dunaliella salina.

The glycerol-3-phosphate dehydrogenase (GPD) gene family plays a major role in glycerol synthesis and adaptation to abiotic stresses. Few studies on GPD family genes from the halotolerant algae Dunaliella salina are available. In this study, seven DsaGPD genes were identified by mining D. salina sequencing data. Among them, DsaGPD5 contained the canonical NAD+-GPD protein domain, called si-GPD. In comparison, DsaGPD1-4 not only contained the canonical NAD+-GPD domain but also a unique domain, the haloacid dehalogenase (HAD)-like superfamily domain, in their N-terminal region, called bi-GPD. DsaGPD6, 7 contained the FAD+-GPD domain. In the transient expression system, DsaGPD1, 3, 4 were found in the cytosol of Arabidopsis thaliana protoplast, DsaGPD2, 5 in the chloroplast, and DsaGPD6, 7 in the mitochondria. MEME analysis showed that six conserved motifs were present in both si-GPDs and bi-GPDs, whereas seven highly conserved motifs were only present in bi-GPDs. The quantitative real-time PCR results showed significant induction of the DsaGPD genes under abiotic stresses, indicating their tolerance-related role in D. salina. DsaGPD2 and DsaGPD5 may be the osmoregulator form and glyceride form in the chloroplast, respectively. The evolutionary forces acting on si-GPDs and bi-GPDs were different in the same organism: bi-GPDs were under purifying selection, while si-GPDs were mainly under positive selection. Furthermore, evolution of the N_HAD domain and C_GPD domain in bi-GPDs is highly correlated. In summary, this study characterizes DsaGPD gene family members and provides useful information for elucidating the salt tolerance mechanism in D. salina.

Effects of triethylamine on the expression patterns of two G3PDHs and lipid accumulation in Dunaliella tertiolecta.

Glycerol-3-phosphate (G3P) is the important precursors for triacylglycerol synthesis, while glycerol-3-phosphate dehydrogenase (GPDH) determines the formation of G3P. In this study, two GDPH genes, Dtgdp1 and Dtgdp2 were isolated and identified from Dunaliella tertiolecta. The full-length Dtgdp1 and Dtgdp2 CDS were 2016 bp and 2094 bp, which encoded two putative protein sequences of 671 and 697 amino acids with predicted molecular weights of 73.64 kDa and 76.73 kDa, respectively. DtGDP1 and DtGDP2 both had a close relationship with those of algal and higher plants. DtGDP1 shared two conserved superfamily (A1 and A2) and four signature motifs (I-IV), and the DtGDP2 showed six signature domains (from motif I to VI) and DAO_C conserved family. Our previous work showed that the triethylamine intervention could greatly increase the triacylglycerol content (up to 80%) of D. tertiolecta. This study aims to investigate the effect of triethylamine on GPDH expression. Results showed that, when treated by triethylamine at 100 ppm and 150 ppm, the expression levels of Dtgdp1 and Dtgpd2 were increased to 5.121- and 56.964-fold compared with the control, respectively. Triethylamine seemed to enhance lipid metabolic flow by inducing the expressions of Dtgdp1 and Dtgdp2 to increase the lipid content, which provides a new insight into the desired pathway of lipid synthesis in algae through genetic engineering.

Human Glycerol 3-Phosphate Dehydrogenase: X-ray Crystal Structures That Guide the Interpretation of Mutagenesis Studies.

Human liver glycerol 3-phosphate dehydrogenase ( hlGPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate, using the binding energy associated with the nonreacting phosphodianion of the substrate to properly orient the enzyme-substrate complex within the active site. Herein, we report the crystal structures for unliganded, binary E·NAD, and ternary E·NAD·DHAP complexes of wild type hlGPDH, illustrating a new position of DHAP, and probe the kinetics of multiple mutant enzymes with natural and truncated substrates. Mutation of Lys120, which is positioned to donate a proton to the carbonyl of DHAP, results in similar increases in the activation barrier to hlGPDH-catlyzed reduction of DHAP and to phosphite dianion-activated reduction of glycolaldehyde, illustrating that these transition states show similar interactions with the cationic K120 side chain. The K120A mutation results in a 5.3 kcal/mol transition state destabilization, and 3.0 kcal/mol of the lost transition state stabilization is rescued by 1.0 M ethylammonium cation. The 6.5 kcal/mol increase in the activation barrier observed for the D260G mutant hlGPDH-catalyzed reaction represents a 3.5 kcal/mol weakening of transition state stabilization by the K120A side chain and a 3.0 kcal/mol weakening of the interactions with other residues. The interactions, at the enzyme active site, between the K120 side chain and the Q295 and R269 side chains were likewise examined by double-mutant analyses. These results provide strong evidence that the enzyme rate acceleration is due mainly or exclusively to transition state stabilization by electrostatic interactions with polar amino acid side chains.

Development of glycerol biosensor based on co-immobilization of enzyme nanoparticles onto graphene oxide nanoparticles decorated pencil graphite electrode.

An improved amperometric biosensor was fabricated by immobilizing glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) nanoparticles (NPs) onto graphene oxide nanoparticles (GrONPs) modified pencil graphite (PG) electrode. The GKNPs, GPONPs and GrONPs were characterized by UV spectroscopy, and transmission electron microscopy (TEM). The working electrode (GKNPs/GPONPs/GrONPs/PGE) was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. The biosensor exhibited optimal current response at an applied potential of 0.45 V, pH 8.0, and 35 °C. The biosensor displayed a wide linear response for glycerol concentration from 0.001 to 60 mM with a detection limit of 0.002 µM. Moreover, a very high sensitivity 121.45 µA·mM-1·cm-2, rapid response time (2 s) and a good concurrence with the standard enzymic colorimetric technique with a correlation coefficient (R2 = 0.99) was offered by the present biosensor. Evidently, biosensor revealed an analytical recovery of 98.5% after addition of glycerol to the sera samples. Within and between batches studies of working electrode demonstrated coefficients of variation of 0.098% and 0.101%, respectively. The biosensor measured blood serum glycerol level in patients suffering from hyperglyceridemia. The biosensor lost 25% of its initial activity after its regular use over a period of 210 days, at 4 °C storage condition.

Fabrication of glycerol biosensor based on co-immobilization of enzyme nanoparticles onto pencil graphite electrode.

Glycerol kinase (GK) and glycerol-3- phosphate oxidase (GPO) nanoparticles (NPs) were prepared, characterized and immobilized onto pencil graphite (PG) electrode to fabricate an improved amperometric glycerol biosensor (GKNPs/GPONPs/PGE). GKNPs/GPONPs/PGE worked in optimum conditions of pH 7.0, temperature 30 °C, at an applied potential of -0.3 V. The biosensor exhibited wide linear response in a concentration range of glycerol (0.01-45 mM) with detection limit 0.0001 µM. The biosensor revealed high sensitivity (7.24 µAmM-1cm-2), low response time (2.5s) and a good agreement with the standard enzymic colorimetric method with a correlation coefficient (R2 = 0.99). The evaluation study of biosensor offered a good analytical recovery of 98.73% when glycerol concentration was added to the sera sample. In addition, within and between batches study of working electrode showed coefficients of variation as 0.105% and 0.14%, respectively. The application of biosensor is performed in the serum of apparently healthy subject and patients affected by cardiogenic shock. There was a 20% loss in initial activity of biosensor after its regular use over a time period of 180 days, while being stored at 4 °C.

Chemical interaction of glycero-phosphate dimethacrylate (GPDM) with hydroxyapatite and dentin.

OBJECTIVES: Although the functional monomer glycero-phosphate dimethacrylate (GPDM) has since long been used in several dental adhesives and more recently in self-adhesive composite cements and restoratives, its mechanism of chemical adhesion to hydroxyapatite (HAp) is still unknown. We therefore investigated the chemical interaction of GPDM with HAp using diverse chemical analyzers and ultra-structurally characterized the interface of a GPDM-based primer formulation with dentin. METHODS: HAp particles were added to a GPDM solution for various periods, upon which they were thoroughly washed with ethanol and water prior to being air-dried. As control, 10-methacryloyloxydecyl dihydrogen phosphate (MDP) was used. The molecular interaction of GPDM with HAp was analyzed using X-ray diffraction (XRD) and solid-state nuclear magnetic resonance (NMR) spectroscopy. Crystal formation upon application of GPDM onto dentin was analyzed using thin-film XRD (TF-XRD). Its hydrophobicity was measured using contact-angle measurement. The interaction of GPDM with dentin was characterized using transmission electron microscopy (TEM). RESULTS: XRD revealed the deposition of dicalcium phosphate dihydrate (DCPD: CaHPO4·2H2O) on HAp after 24h. NMR confirmed the adsorption of GPDM onto HAp. However, GPDM was easily removed after washing with water, unlike MDP that remained adhered to HAp. Dentin treated with GPDM appeared more hydrophilic compared to dentin treated with MDP. TEM disclosed exposed collagen in the hybrid layer produced by the GPDM-based primer formulation. SIGNIFICANCE: Although GPDM adsorbed to HAp, it did not form a stable calcium salt. The bond between GPDM and HAp was weak, unlike the strong bond formed by MDP to HAp. Due to its high hydrophilicity, GPDM might be an adequate monomer for an etch-and-rinse adhesive, but appears less appropriate for a 'mild' self-etch adhesive that besides micro-retention ionically interacts with HAp, or for a self-adhesive restorative material.

Etching Efficacy of Self-Etching Functional Monomers.

Besides chemically interacting with hard tooth tissue, acidic functional monomers of self-etch adhesives should etch the prepared tooth surface to dissolve the smear layer and to provide surface micro-retention. Although the etching efficacy of functional monomers is commonly determined in terms of pH, the pH of adhesives cannot accurately be measured. Better is to measure the hydroxyapatite (HAp)-dissolving capacity, also considering that functional monomers may form monomer-Ca salts. Here, the etching efficacy of 6 functional monomers (GPDM, phenyl-P, MTEGP, 4-META, 6-MHP and 10-MDP) was investigated. Solutions containing 15 wt% monomer, 45 wt% ethanol, and 40 wt% water were prepared. Initially, we observed enamel surfaces exposed to monomer solution by scanning electron microscopy (SEM). X-ray diffraction (XRD) was employed to detect monomer-Ca salt formation. Phenyl-P exhibited a strong etching effect, while 10-MDP-treated enamel showed substance deposition, which was identified by XRD as 10-MDP-Ca salt. To confirm these SEM/XRD findings, we determined the etching efficacy of functional monomers by measuring both the concentration of Ca released from HAp using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and the amount of monomer-Ca salt formation using 31P magic-angle spinning (MAS) nuclear magnetic resonance (NMR). ICP-AES revealed that the highest Ca concentration was produced by phenyl-P and the lowest Ca concentration, almost equally, by 4-META and 10-MDP. Only 10-MDP formed 10-MDP-Ca salts, indicating that 10-MDP released more Ca from HAp than was measured by ICP-AES. Part of the released Ca was consumed to form 10-MDP-Ca salts. It is concluded that the repeatedly reported higher bonding effectiveness of 10-MDP-based adhesives must not only be attributed to the more intense chemical bonding of 10-MDP but also to its higher etching potential, a combination the other functional monomers investigated lack.

Enzyme Architecture: The Role of a Flexible Loop in Activation of Glycerol-3-phosphate Dehydrogenase for Catalysis of Hydride Transfer.

The side chain of Q295 of glycerol-3-phosphate dehydrogenase from human liver ( hlGPDH) lies in a flexible loop, that folds over the phosphodianion of substrate dihydroxyacetone phosphate (DHAP). Q295 interacts with the side-chain cation from R269, which is ion-paired to the substrate phosphodianion. Kinetic parameters kcat/ Km (M-1 s-1) and kcat/ KGA KHPi (M-2 s-1) were determined, respectively, for catalysis of the reduction of DHAP and for dianion activation of catalysis of reduction of glycolaldehyde (GA) catalyzed by wild-type, Q295G, Q295S, Q295A, and Q295N mutants of hlGPDH. These mutations result in up to a 150-fold decrease in ( kcat/ Km)DHAP and up to a 2.7 kcal/mol decrease in the intrinsic phosphodianion binding energy. The data define a linear correlation with slope 1.1, between the intrinsic phosphodianion binding energy and the intrinsic phosphite dianion binding energy for activation of hlGPDH-catalyzed reduction of GA, that demonstrates a role for Q295 in optimizing this dianion binding energy. The R269A mutation of wild-type GPDH results in a 9.1 kcal/mol destabilization of the transition state for reduction of DHAP, but the same R269A mutation of N270A and Q295A mutants result in smaller 5.9 and 4.9 kcal/mol transition-state destabilization. Similarly, the N270A or Q295A mutations of R269A GPDH each result in large falloffs in the efficiency of rescue of the R269A mutant by guanidine cation. We conclude that N270, which interacts for the substrate phosphodianion and Q295, which interacts with the guanidine side chain of R269, function to optimize the apparent transition-state stabilization provided by the cationic side chain of R269.

A reevaluation of the origin of the rate acceleration for enzyme-catalyzed hydride transfer.

There is no consensus of opinion on the origin of the large rate accelerations observed for enzyme-catalyzed hydride transfer. The interpretation of recent results from studies on hydride transfer reactions catalyzed by alcohol dehydrogenase (ADH) focus on the proposal that the effective barrier height is reduced by quantum-mechanical tunneling through the energy barrier. This interpretation contrasts sharply with the notion that enzymatic rate accelerations are obtained through direct stabilization of the transition state for the nonenzymatic reaction in water. The binding energy of the dianion of substrate DHAP provides 11 kcal mol-1 stabilization of the transition state for the hydride transfer reaction catalyzed by glycerol-3-phosphate dehydrogenase (GPDH). We summarize evidence that the binding interactions between (GPDH) and dianion activators are utilized directly for stabilization of the transition state for enzyme-catalyzed hydride transfer. The possibility is considered, and then discounted, that these dianion binding interactions are utilized for the stabilization of a tunnel ready state (TRS) that enables efficient tunneling of the transferred hydride through the energy barrier, and underneath the energy maximum for the transition state. It is noted that the evidence to support the existence of a tunnel-ready state for the hydride transfer reactions catalyzed by ADH is ambiguous. We propose that the rate acceleration for ADH is due to the utilization of the binding energy of the cofactor NAD+/NADH in the stabilization of the transition state for enzyme-catalyzed hydride transfer.

Pnc1 piggy-back import into peroxisomes relies on Gpd1 homodimerisation.

Peroxisomes are eukaryotic organelles that posttranslationally import proteins via one of two conserved peroxisomal targeting signal (PTS1 or 2) mediated pathways. Oligomeric proteins can be imported via these pathways but evidence is accumulating that at least some PTS1-containing monomers enter peroxisomes before they assemble into oligomers. Some proteins lacking a PTS are imported by piggy-backing onto PTS-containing proteins. One of these proteins is the nicotinamidase Pnc1, that is co-imported with the PTS2-containing enzyme Glycerol-3-phosphate dehydrogenase 1, Gpd1. Here we show that Pnc1 co-import requires Gpd1 to form homodimers. A mutation that interferes with Gpd1 homodimerisation does not prevent Gpd1 import but prevents Pnc1 co-import. A suppressor mutation that restores Gpd1 homodimerisation also restores Pnc1 co-import. In line with this, Pnc1 interacts with Gpd1 in vivo only when Gpd1 can form dimers. Redirection of Gpd1 from the PTS2 import pathway to the PTS1 import pathway supports Gpd1 monomer import but not Gpd1 homodimer import and Pnc1 co-import. Our results support a model whereby Gpd1 may be imported as a monomer or a dimer but only the Gpd1 dimer facilitates co-transport of Pnc1 into peroxisomes.

Enzyme activation through the utilization of intrinsic dianion binding energy.

43: We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed.

Amperometric triglyceride bionanosensor based on nanoparticles of lipase, glycerol kinase, glycerol-3-phosphate oxidase.

The nanoparticles (NPs) aggregates of lipase from porcine pancreas, glycerol kinase (GK) from Cellulomonas sp. and glycerol-3-phosphate oxidase (GPO) from Aerococcus viridanss were prepared by desolvation and glutaraldehyde crosslinking and functionalized by cysteamine. These enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM) and Fourier transform infra red (FTIR) spectroscopy. The functionalzed ENPs aggregates were co-immobilized covalently onto polycrystalline Au electrode through thiolated bond. An improved amperometric triglyceride (TG) bionanosensor was constructed using this ENPs modified Au electrode as working electrode. Biosensor showed optimum current at 1.2 V within 5s, at pH 6.5 and 35 °C.A linear relationship was obtained between current (mA) and triolein concentration in lower concentration range,10-100 mg/dL and higher concentration range, 100-500 mg/dL. Limit of detection (LOD) of bionanosensor was 1.0 µg/ml. Percent analytical recovery of added trolein (50 and 100 mg/dL) in serum was 95.2 ± 0.5 and 96.0 ± 0.17. Within and between batch coefficients of variation (CV) were 2.33% and 2.15% respectively. A good correlation (R2 = 0.99) was obtained between TG values in sera measured by present biosensor and standard enzymic colorimetric method with the regression equation: y= (0.993x + 0.967). ENPs/Au electrode was used 180 times over a period of 3 months with 50% loss in its initial activity, when stored dry at 4 °C.

Structure-Reactivity Effects on Intrinsic Primary Kinetic Isotope Effects for Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase.

Primary deuterium kinetic isotope effects (1°DKIE) on (kcat/KGA, M-1 s-1) for dianion (X2-) activated hydride transfer from NADL to glycolaldehyde (GA) catalyzed by glycerol-3-phosphate dehydrogenase were determined over a 2100-fold range of enzyme reactivity: (X2-, 1°DKIE); FPO32-, 2.8 ± 0.1; HPO32-, 2.5 ± 0.1; SO42-, 2.8 ± 0.2; HOPO32-, 2.5 ± 0.1; S2O32-, 2.9 ± 0.1; unactivated; 2.4 ± 0.2. Similar 1°DKIEs were determined for kcat. The observed 1°DKIEs are essentially independent of changes in enzyme reactivity with changing dianion activator. The results are consistent with (i) fast and reversible ligand binding; (ii) the conclusion that the observed 1°DKIEs are equal to the intrinsic 1°DKIE on hydride transfer from NADL to GA; (iii) similar intrinsic 1°DKIEs on GPDH-catalyzed reduction of the substrate pieces and the whole physiological substrate dihydroxyacetone phosphate. The ground-state binding interactions for different X2- are similar, but there are large differences in the transition state interactions for different X2-. The changes in transition state binding interactions are expressed as changes in kcat and are proposed to represent changes in stabilization of the active closed form of GPDH. The 1°DKIEs are much smaller than observed for enzyme-catalyzed hydrogen transfer that occurs mainly by quantum-mechanical tunneling.

Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer.

The stabilization of the transition state for hlGPDH-catalyzed reduction of DHAP due to the action of the phosphodianion of DHAP and the cationic side chain of R269 is between 12.4 and 17 kcal/mol. The R269A mutation of glycerol-3-phosphate dehydrogenase (hlGPDH) results in a 9.1 kcal/mol destabilization of the transition state for enzyme-catalyzed reduction of dihydroxyacetone phosphate (DHAP) by NADH, and there is a 6.7 kcal/mol stabilization of this transition state by 1.0 M guanidine cation (Gua+) [J. Am. Chem. Soc. 2015, 137, 5312-5315]. The R269A mutant shows no detectable activity toward reduction of glycolaldehyde (GA), or activation of this reaction by 30 mM HPO32-. We report the unprecedented self-assembly of R269A hlGPDH, dianions (X2- = FPO32-, HPO32-, or SO42-), Gua+ and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants kcat/KGAKXKGua. The linear logarithmic correlation (slope = 1.0) between values of kcat/KGAKX for dianion activation of wildtype hlGPDH-catalyzed reduction of GA and kcat/KGAKXKGua shows that the electrostatic interaction between exogenous dianions and the side chain of R269 is not significantly perturbed by cutting hlGPDH into R269A and Gua+ pieces. The advantage for connection of hlGPDH (R269A mutant + Gua+) and substrate pieces (GA + HPi) pieces, (ΔGS‡)HPi+E+Gua = 5.6 kcal/mol, is nearly equal to the sum of the advantage to connection of the substrate pieces, (ΔGS‡)GA+HPi = 3.3 kcal/mol, for wildtype hlGPDH-catalyzed reaction of GA + HPi, and for connection of the enzyme pieces, (ΔGS‡)E+Gua = 2.4 kcal/mol, for Gua+ activation of the R269A hlGPDH-catalyzed reaction of DHAP.