Altered Enzyme Kinetics Research Articles

Redox potentials elucidate the electron transfer pathway of NAD+-dependent formate dehydrogenases

Metal-dependent, nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs) are complex metalloenzymes coupling biochemical transformations through intricate electron transfer pathways. Rhodobacter capsulatus FDH is a model enzyme for understanding coupled catalysis, in that reversible CO2 reduction and formate oxidation are linked to a flavin mononuclotide (FMN)-bound diaphorase module via seven iron-sulfur (FeS) clusters as a dimer of heterotetramers. Catalysis occurs at a bis-metal-binding pterin (Mo) binding two molybdopterin guanine dinucleotides (bis-MGD), a protein-based Cys residue and a participatory sulfido ligand. Insights regarding the proposed electron transfer mechanism between the bis-MGD and the FMN have been complicated by the discovery that an alternative pathway might occur via intersubunit electron transfer between two [4Fe4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the FeS clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven FeS clusters could be assigned. Furthermore, substitution of the active site residue Lys295 with Ala resulted in altered enzyme kinetics, primarily due to a more negative redox potential of the A1 [4Fe4S] cluster. Finally, characterization of the monomeric FdsGBAD heterotetramer exhibited slightly decreased formate oxidation activity and similar iron-sulfur clusters reduced relative to the dimeric heterotetramer. Comparison of the measured redox potentials relative to structurally defined FeS clusters support a mechanism by which electron transfer occurs within a heterotetrameric unit, with the interfacial [4Fe4S] cluster serving as a structural component toward the integrity of the heterodimeric structure to drive efficient catalysis.

Dynamic Domain Links Substrate Binding and Catalysis in the Factor-Inhibiting-HIF-1.

The interplay between active-site chemistry and functionally relevant enzyme motions can provide useful insights into selective enzyme modulation. Modulation of the hypoxia-sensing function of factor-inhibiting-HIF-1 (FIH) enzyme is a potential therapeutic strategy in disease states such as ischemia and cancer. The hypoxia-sensing function of FIH relies in major part on the tight coupling of the first half of the catalytic mechanism which involves O2 activation and eventual succinate production to the second half which involves HIF-1α/CTAD substrate hydroxylation. In this study, we demonstrate the role of a loop hinge domain in FIH (FIH102-118) called the 100s loop in maintaining this particular tight coupling. Molecular dynamics patterns from Gaussian Network Model (iGNM) database analysis of FIH identified the 100s loop as one dynamic domain containing a hinge residue (Tyr102) with a potential substrate positioning role. Enzymological and biophysical studies of the 100s loop point mutants revealed altered enzyme kinetics with the exception of the conservative FIH mutant Y102F, which suggests a sterics-related role for this residue. Removal of the bulk of Tyr102 (Y102A) resulted in succinate production, autohydroxylation, and an O2 binding environment comparable to wild-type FIH. However, the HIF-1α/CTAD substrate hydroxylation of this mutant was significantly reduced which implies that (1) the FIH loop hinge residue Tyr102 does not affect O2 activation, (2) the stacking steric interaction of Tyr102 is important in substrate positioning for productive hydroxylation, and (3) Tyr102 is important for the synchronization of O2 activation and substrate hydroxylation.

Heme-Edge Residues Modulate Signal Transduction within a Bifunctional Homo-Dimeric Sensor Protein.

Bifunctional enzymes, which contain two domains with opposing enzymatic activities, are widely distributed in bacteria, but the regulatory mechanism(s) that prevent futile cycling are still poorly understood. The recently described bifunctional enzyme, DcpG, exhibits unusual heme properties and is surprisingly able to differentially regulate its two cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic domains in response to heme gaseous ligands. Mutagenesis of heme-edge residues was used to probe the heme pocket and resulted in decreased O2 dissociation kinetics, identifying roles for these residues in modulating DcpG gas sensing. In addition, the resonance Raman spectra of the DcpG wild type and heme-edge mutants revealed that the mutations alter the heme electrostatic environment, vinyl group conformations, and spin state population. Using small-angle X-ray scattering and negative stain electron microscopy, the heme-edge mutations were demonstrated to cause changes to the protein conformation, which resulted in altered signaling transduction and enzyme kinetics. These findings provide insights into molecular interactions that regulate DcpG gas sensing as well as mechanisms that have evolved to control multidomain bacterial signaling proteins.

Phthalate hydrolase: distribution, diversity and molecular evolution.

The alpha/beta-fold superfamily of hydrolases is rapidly becoming one of the largest groups of structurally related enzymes with diverse catalytic functions. In this superfamily of enzymes, esterase deserves special attention because of their wide distribution in biological systems and importance towards environmental and industrial applications. Among various esterases, phthalate hydrolases are the key alpha/beta enzymes involved in the metabolism of structurally diverse estrogenic phthalic acid esters, ubiquitously distributed synthetic chemicals, used as plasticizer in plastic manufacturing processes. Although they vary both at the sequence and functional levels, these hydrolases use a similar acid-base-nucleophile catalytic mechanism to catalyse reactions on structurally different substrates. The current review attempts to present insights on phthalate hydrolases, describing their sources, structural diversities, phylogenetic affiliations and catalytically different types or classes of enzymes, categorized as diesterase, monoesterase and diesterase-monoesterase, capable of hydrolysing phthalate diester, phthalate monoester and both respectively. Furthermore, available information on in silico analyses and site-directed mutagenesis studies revealing structure-function integrity and altered enzyme kinetics have been highlighted along with the possible scenario of their evolution at the molecular level.

Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production

Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure. Instead, we show that cyclin-dependent kinases (CDKs) are activated by the HFD and responsible for PKL phosphorylation at position S113 in addition to other targets. These CDKs control PKL nuclear retention, alter cytosolic PKL activity, and ultimately influence glucose production. These results change our view of PKL regulation and highlight a previously unrecognized pathway of hepatic CDK activity and metabolic control points that may be important in insulin resistance and type 2 diabetes.

Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)

Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a ~45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM.Graphical ᅟ

Genetic variants in ALDH1B1 and alcohol dependence risk in a British and Irish population: A bioinformatic and genetic study.

Alcohol is metabolized in the liver via the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Polymorphisms in the genes encoding these enzymes, which are common in East Asian populations, can alter enzyme kinetics and hence the risk of alcohol dependence and its sequelae. One of the most important genetic variants, in this regards, is the single nucleotide polymorphism (SNP) rs671 in ALDH2, the gene encoding the primary acetaldehyde metabolizing enzyme ALDH2. However, the protective allele of rs671 is absent in most Europeans although ALDH1B1, which shares significant sequence homology with ALDH2, contains several, potentially functional, missense SNPs that do occur in European populations. The aims of this study were: (i) to use bioinformatic techniques to characterize the possible effects of selected variants in ALDH1B1 on protein structure and function; and, (ii) to genotype three missense and one stop-gain, protein-altering, non-synonymous SNPs in 1478 alcohol dependent cases and 1254 controls of matched British and Irish ancestry. No significant allelic associations were observed between the three missense SNPs and alcohol dependence risk. The minor allele frequency of rs142427338 (Gln378Ter) was higher in alcohol dependent cases than in controls (allelic P = 0.19, OR = 2.98, [0.62–14.37]) but as this SNP is very rare the study was likely underpowered to detect an association with alcohol dependence risk. This potential association will needs to be further evaluated in other large, independent European populations.

Regulation of Cytochrome C by Phosphorylation: Mitochondrial Respiration and Apoptosis

Mammalian cytochrome c (Cytc) is a small globular protein and functions as a mobile electron carrier in the electron transport chain (ETC), shuttling electrons from bc1 complex to cytochrome c oxidase (COX). In addition, Cytc participates in type II apoptosis, when it is released from the mitochondria. Such a molecule, central to life and death decisions of the cell, should be tightly controlled. We previously showed that Cytc is regulated by cell signaling pathways in a highly tissue-specific manner and that Cytc isolated from heart and liver is phosphorylated on Tyr97 and Tyr48, respectively. Here we add another phosphorylation site and show that Cytc isolated from kidney is phosphorylated on Thr28. This phosphorylation is likely mediated by AMP kinase. Phosphorylated and Thr28Glu phosphomimetic Cytc cause partial inhibition in the reaction with COX supporting our model that under healthy conditions ETC proteins are phosphorylated to limit electron flux in the electron transport chain, which in turn prevents a hyperpolarization of the mitochondrial membrane potential, a known cause of reactive oxygen species (ROS) production and trigger of apoptosis. To test our model we generated Cytc knockout cells expressing Thr28Glu phosphomimetic Cytc. We observed reduction of intact cellular respiration, mitochondrial membrane potential, and ROS. Finally, high resolution crystal structure analyses and molecular dynamics simulations suggest that the Thr28-containing epitope is the most flexible internal part of Cytc, which is in close proximity to key residues on COX and thus suggests a mechanism for the altered enzyme kinetics.

Slowed Diffusion and Excluded Volume Both Contribute to the Effects of Macromolecular Crowding on Alcohol Dehydrogenase Steady-State Kinetics.

To understand the consequences of macromolecular crowding, studies have largely employed in vitro experiments with synthetic polymers assumed to be both pure and "inert". These polymers alter enzyme kinetics by excluding volume that would otherwise be available to the enzymes, substrates, and products. Presented here is evidence that other factors, in addition to excluded volume, must be considered in the interpretation of crowding studies with synthetic polymers. Dextran has a weaker effect on the Michaelis-Menten kinetic parameters of yeast alcohol dehydrogenase (YADH) than its small molecule counterpart, glucose. For glucose, the decreased Vmax values directly correlate with slower translational diffusion and the decreased Km values likely result from enhanced substrate binding due to YADH stabilization. Because dextran is unable to stabilize YADH to the same extent as glucose, this polymer's ability to decrease Km is potentially due to the nonideality of the solution, a crowding-induced conformational change, or both. Chronoamperometry reveals that glucose and dextran have surprisingly similar ferricyanide diffusion coefficients. Thus, the reduction in Vmax values for glucose is partially offset by an additional macromolecular crowding effect with dextran. Finally, this is the first report that supplier-dependent impurities in dextran affect the kinetic parameters of YADH. Taken together, our results reveal that caution should be used when interpreting results obtained with inert synthetic polymeric agents, as additional effects from the underlying monomer need to be considered.

Tuning Enzyme Kinetics through Designed Intermolecular Interactions Far from the Active Site

Enzyme–DNA nanostructures were designed to introduce new substrate–enzyme interactions into their reactions, which altered enzyme kinetics in a predictable manner. The designed enzymes demonstrate a new strategy of enzyme engineering based on the rational design of intermolecular interactions outside of the active site that enhance and control enzyme kinetics. Binding interactions between tetramethylbenzidine and DNA attached to horseradish peroxidase (HRP) resulted in a reduced Michaelis constant (KM) for the substrate. The enhancement increased with stronger interactions in the micromolar range, resulting in a 2.6 fold increase in kcat/KM. The inhibition effect of 4-nitrobenzoic hydrazide on HRP was also significantly enhanced by tuning the binding to HRP–DNA. Lastly, binding of a nicotinamide adenine dinucleotide (NAD(H)) cofactor mimic, nicotinamide mononucleotide (NMN(H)), to an aldo-keto reductase (AdhD) was enhanced by introducing NMN(H)–DNA interactions.

Monitoring phospholipase A₂ activity with Gd-encapsulated phospholipid liposomes.

To date, numerous analytical methods have been developed to monitor phospholipase A2 (PLA2) activity. However, many of these methods require the use of unnatural PLA2 substrates that may alter enzyme kinetics, and probes that cannot be extended to applications in more complex environments. It would be desirable to develop a versatile assay that monitors PLA2 activity based on interactions with natural phospholipids in complex biological samples. Here, we developed an activatable T1 magnetic resonance (MR) imaging contrast agent to monitor PLA2 activity. Specifically, the clinically approved gadolinium (Gd)-based MR contrast agent, gadoteridol, was encapsulated within nanometer-sized phospholipid liposomes. The encapsulated Gd exhibited a low T1-weighted signal, due to low membrane permeability. However, when the phospholipids within the liposomal membrane were hydrolyzed by PLA2, encapsulated Gd was released into bulk solution, resulting in a measureable change in the T1-relaxation time. These activatable MR contrast agents can potentially be used as nanosensors for monitoring of PLA2 activity in biological samples with minimal sample preparation.

Backbone 1H, 13C, and 15N chemical shift assignment for HIV-1 protease subtypes and multi-drug resistant variant MDR 769

HIV-1 protease (HIV-1PR) is an essential drug target in the treatment of patients infected with HIV-1. Mutations are found to arise in over 38 of 99 amino acid sites in this protein in response to drug therapy or natural selection, where many are found combinations that alter enzyme kinetics or inhibitor susceptibility without a clear structural mechanism. In efforts to understand how these mutations alter the flexibility and dynamics of HIV-1PR, we report the backbone (1)H, (13)C, and (15)N chemical shift assignments for subtypes C, circulating recombinant form CRF01_AE and a multi-drug resistant variant MDR 769. These assignments are essential for future work aimed at characterizing backbone dynamics, exchange dynamics and dynamics of protein/substrate or protein/inhibitor interactions.

Effects of single-nucleotide polymorphisms in the human holocarboxylase synthetase gene on enzyme catalysis

Holocarboxylase synthetase (HLCS) is a biotin protein ligase, which has a pivotal role in biotin-dependent metabolic pathways and epigenetic phenomena in humans. Knockdown of HLCS produces phenotypes such as heat susceptibility and decreased life span in Drosophila melanogaster, whereas knockout of HLCS appears to be embryonic lethal. HLCS comprises 726 amino acids in four domains. More than 2500 single-nucleotide polymorphisms (SNPs) have been identified in human HLCS. Here, we tested the hypotheses that HLCS SNPs impair enzyme activity, and that biotin supplementation restores the activities of HLCS variants to wild-type levels. We used an in silico approach to identify five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96 L SNP did not alter enzyme kinetics. Our findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.

Urate nephropathy associated with impaired kinetic properties of hypoxanthine phosphoribosyl transferase in a 45-day-old infant

We report a 45-day-old male infant who presented with anuric renal failure and fluid overload due to urate nephropathy consequent upon hyperuricemia with hyperuricosuria. His maternal uncle had undergone renal transplantation for chronic renal failure secondary to uric acid nephrolithiasis. The levels of hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase activity in the baby were found to be quantitatively normal. However, when the HPRT activity was measured at low substrate concentrations [phosphoribosyl pyrophosphate (PRPP) and hypoxanthine]and compared with usual assay conditions, the HPRT activity at lower PRPP was less in the propositus, suggesting altered enzyme kinetics. Apparent K (m(PRPP)) and V (max), but not K (m(hypoxanthine)), were then found to be higher in the propositus than the control range. This is the first case of urate nephropathy secondary to altered enzyme kinetics presenting as early as 45days. Uric acid nephropathy should be considered in the differential diagnosis of unexplained acute kidney injury in infants. In such cases, quantitative tests for HPRT enzyme activity may not be sufficient and altered enzyme kinetics should also be investigated.

The Tail Wagging the Dog: Insights into Catalysis in R67 Dihydrofolate Reductase

Plasmid-encoded R67 dihydrofolate reductase (DHFR) catalyzes a hydride transfer reaction between substrate dihydrofolate (DHF) and its cofactor, nicotinamide adenine dinucleotide phosphate (NADPH). R67 DHFR is a homotetramer that exhibits numerous characteristics of a primitive enzyme, including promiscuity in binding of substrate and cofactor, formation of nonproductive complexes, and the absence of a conserved acid in its active site. Furthermore, R67's active site is a pore, which is mostly accessible by bulk solvent. This study uses a computational approach to characterize the mechanism of hydride transfer. Not surprisingly, NADPH remains fixed in one-half of the active site pore using numerous interactions with R67. Also, stacking between the nicotinamide ring of the cofactor and the pteridine ring of the substrate, DHF, at the hourglass center of the pore, holds the reactants in place. However, large movements of the p-aminobenzoylglutamate tail of DHF occur in the other half of the pore because of ion pair switching between symmetry-related K32 residues from two subunits. This computational result is supported by experimental results that the loss of these ion pair interactions (located >13 Å from the center of the pore) by addition of salt or in asymmetric K32M mutants leads to altered enzyme kinetics [Hicks, S. N., et al. (2003) Biochemistry 42, 10569-10578; Hicks, S. N., et al. (2004) J. Biol. Chem. 279, 46995-47002]. The tail movement at the edge of the active site, coupled with the fixed position of the pteridine ring in the center of the pore, leads to puckering of the pteridine ring and promotes formation of the transition state. Flexibility coupled to R67 function is unusual as it contrasts with the paradigm that enzymes use increased rigidity to facilitate attainment of their transition states. A comparison with chromosomal DHFR indicates a number of similarities, including puckering of the nicotinamide ring and changes in the DHF tail angle, accomplished by different elements of the dissimilar protein folds.

Finger Domain Mutation Affects Enzyme Activity, DNA Replication Efficiency, and Fidelity of an Exonuclease-Deficient DNA Polymerase of Herpes Simplex Virus Type 1

The catalytic subunit of herpes simplex virus DNA polymerase (Pol), a member of the B family polymerases, possesses both polymerase and exonuclease activities. We previously demonstrated that a recombinant virus (YD12) containing a double mutation within conserved exonuclease motif III of the Pol was highly mutagenic and rapidly evolved to contain an additional leucine-to-phenylalanine mutation at residue 774 (L774F), which is located within the finger subdomain of the polymerase domain. We further demonstrated that the recombinant L774F virus replicated DNA with increased fidelity and that the L774F mutant Pol exhibited altered enzyme kinetics and impaired polymerase activity to extension from mismatched primer termini. In this study, we demonstrated that addition of the L774F mutation to the YD12 Pol did not restore the exonuclease deficiency. However, the polymerase activity of the YD12 Pol to extension from mismatched primer termini and on the nucleotide incorporation pattern was altered upon addition of the L774F mutation. The L774F mutation-containing YD12 Pol also supported the growth of viral progeny and replicated DNA more efficiently and more accurately than did the YD12 Pol. Together, these studies demonstrate that a herpes simplex virus Pol mutant with a highly mutagenic ability can rapidly acquire additional mutations, which may be selected for their survival and outgrowth. Furthermore, the studies demonstrate that the polymerase activity of HSV-1 Pol on primer extension is influenced by sequence context and that herpes simplex virus type 1 Pol may dissociate more frequently at G.C sites during the polymerization reaction. The implications of the findings are discussed.

Novel mutations in 3-phosphoglycerate dehydrogenase (PHGDH) are distributed throughout the protein and result in altered enzyme kinetics

Three-phosphoglycerate dehydrogenase (3-PGDH) deficiency is a rare recessive inborn error in the biosynthesis of the amino acid L-serine characterized clinically by congenital microcephaly, psychomotor retardation, and intractable seizures. The biochemical abnormalities associated with this disorder are low concentrations of L-serine, D-serine, and glycine in cerebrospinal fluid (CSF). Only two missense mutations (p.V425M and p.V490M) have been identified in PHGDH, the gene encoding 3-PGDH, but it is currently unclear how these mutations in the carboxy-terminal regulatory domain of the protein affect enzyme function. We now describe five novel mutations in five patients with 3-PGDH deficiency; one frameshift mutation (p.G238fsX), and four missense mutations (p.R135W, p.V261M, p.A373T, and p.G377S). The missense mutations were located in the nucleotide binding and regulatory domains of 3-PGDH and did not affect steady-state expression, protein stability, and protein degradation rates. Patients' fibroblasts displayed a significant, but incomplete, reduction in maximal enzyme activities associated with all missense mutations. In transient overexpression studies in HEK293T cells, the p.A373T, p.V425M, and p.V490M mutations resulted in almost undetectable enzyme activities. Molecular modeling of the p.R135W and p.V261M mutations onto the partial crystal structure of 3-PGDH predicted that these mutations affect substrate and cofactor binding. This prediction was confirmed by the results of kinetic measurements in fibroblasts and transiently transfected HEK293T cells, which revealed a markedly decreased V(max) and an increase in K(m) values, respectively. Taken together, these data suggest that missense mutations associated with 3-PGDH deficiency either primarily affect substrate binding or result in very low residual enzymatic activity.

Effect of CYP2A13 Active Site Mutation N297A on Metabolism of Coumarin and Tobacco-Specific Nitrosamines

Cytochrome P450 2A13-catalyzed alpha-hydroxylation is a critical step in the activation of the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and (S)-N'-nitrosonornicotine [(S)-NNN]. In the enzyme's active site, a single polar residue, Asn297, can influence substrate binding, orientation, and metabolism. We determined the effects of N297A mutation on enzyme kinetics and specificity for NNK, NNN, and coumarin metabolism. [5-(3)H]-NNK, [5-(3)H]-(S)-NNN, [(14)C]coumarin, and radioflow high-performance liquid chromatography analysis were used to quantify metabolites. Cytochrome P450 (P450) 2A13 N297A catalyzed NNK alpha-hydroxylation, with a 3-fold preference for methylene versus methyl hydroxylation, similar to wild type. Docking studies using the P450 2A13 crystal structure predicted that when the pyridine ring of NNK cannot hydrogen bond to residue 297 it tilts and orients NNK in positions unfavorable for alpha-hydroxylation. The N297A mutation resulted in a 5- and 4-fold decrease in catalytic efficiency of NNK and NNN metabolism, respectively, primarily because of increased K(m) values. The N297A mutation strikingly affected coumarin metabolism. The ratio of coumarin 7-hydroxylation to coumarin 3,4-epoxidation is approximately equal for wild-type enzyme, whereas the ratio was 1:9 for the N297A mutant. Coumarin 3,4-epoxidation was significantly underestimated unless the epoxide was trapped and quantified as its glutathione conjugate. The K(m) value for this reaction was 4-fold greater for the mutant enzyme; the V(max) value increased nearly 40-fold. The observed shift toward coumarin 3,4-epoxidation is consistent with docking studies. In summary, Asn297 in P450 2A13 is important for orienting NNK and coumarin in the active site, changing this residue to Ala results in altered enzyme kinetics for NNK, NNN, and coumarin.

Cell Biology Assessment of Glucokinase Mutations V62M and G72R in Pancreatic β-Cells

Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis. In some cases (V62M and G72R), the phenotype cannot be explained by altered enzyme kinetics or protein instability. We used transient and stable expression of green fluorescent protein (GFP) GK chimaeras in MIN6 beta-cells to study the phenotype defect of V62M and G72R. GK activity in lysates of MIN6 cell lines stably expressing wild-type or mutant GFP GK showed the expected affinity for glucose and response to pharmacological activators, indicating the expression of catalytically active enzymes. MIN6 cells stably expressing GFP V62M or GFP G72R had a lower GK activity-to-GK immunoreactivity ratio and GK activity-to-GK mRNA ratio but not GK immunoreactivity-to-GK mRNA ratio than wild-type GFP GK. Heterologous expression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FDP2) in cell lines increased GK activity for wild-type GK and V62M but not for G72R, whereas expression of liver GK regulatory protein (GKRP) increased GK activity for wild type but not V62M or G72R. Lack of interaction of these mutants with GKRP was also evident in hepatocyte transfections from the lack of nuclear accumulation. These results suggest that cellular loss of GK catalytic activity rather than impaired translation or enhanced protein degradation may account for the hyperglycemia in subjects with V62M and G72R mutations.

Altered β-secretase enzyme kinetics and levels of both BACE1 and BACE2 in the Alzheimer’s disease brain

β-Secretase is the rate limiting enzymatic activity in the production of amyloid-β peptide, the primary component of senile plaque pathology in Alzheimer’s disease (AD). This study performed the first comparative analysis of β-secretase enzyme kinetics in AD and control brain tissue. Results found V max values for β-secretase to be significantly increased, and K m values unchanged in AD temporal cortex compared to matched control temporal cortex. The increased V max in AD cases, did not correlate with levels of BACE1, and decreased BACE1 and BACE2 levels correlated with the severity of neurofibrillary pathology (I–VI), and synaptic loss in AD. These results indicate that increased V max for β-secretase is a feature of AD pathogenesis and this increase does not correlate directly with levels of BACE1, the principal β-secretase in brain.