Catalytic Inefficiency Research Articles

UV-assisted photochemical transformation of a tetranuclear copper(II) complex: a DFT supported study on β-lactamase inhibitory activity towards antibiotic resistance.

Herein, we present a dark-green crystalline tetranuclear Cu(II) Schiff base complex {C1 = [Cu4L4](ClO4)4(DMF)4(H2O)} using a N,N,O donor ligand (HL), namely 2-(((2-hydroxypropyl)imino)methyl)-6-methoxyphenol. Spectro-photometrical investigation on the β-lactamase-like activity of this coordinately saturated system revealed its catalytic inefficiency towards hydrolysis of nitrocefin as a model substrate. This complex has attracted significant interest as a promising photo-catalyst owing to its narrow band gap (2.40 eV) as predicted from DFT calculations and its higher responsivity towards UV light. Therefore, C1 is effectively involved in the photocatalytic reduction of perchlorate to Cl- in the presence of a hole scavenger (H2O-MeOH) under prolonged UV irradiation and itself becomes photo-cleaved to yield a new dark-brown colored chlorobridged dinuclear crystalline complex C2 {[CuL(H2O)2Cl3]H2O}. Furthermore, C2 was deployed as a functional β-lactamase model and was found to show a remarkable catalytic proficiency towards the hydrolysis of nitrocefin in 70 : 30 (V/V) MeOH-H2O medium. This pro-catalyst C2 has been speculated to generate an aqua bridged active catalyst that plays a crucial factor in hydrolysis. This phenomenon was again experimentally established by potentiometric pH titration where C2 displays only one pKa value (7.11) in the basic pH range, indicating the deprotonation of the bridged water molecule. Based on several other kinetic studies, it may be postulated that the hydrolysis of nitrocefin is initiated by the nucleophilic attack of a bridging hydroxide, followed by very fast protonation of the intermediate to furnish the hydrolyzed product. It is noteworthy that the rate of nitrocefin hydrolysis is greatly inhibited in the presence of external chloride concentration. To the best of our knowledge, this is the first report on the photochemical behavior of such a tetranuclear copper(II) Schiff base complex. Our current interest is focused on inventing a potent β-lactamase inhibitory therapeutic as well as elucidating its mechanism through comprehensive chemical analysis.

Dual isolated bimetal single-atom catalysts for tumor ROS cycle and parallel catalytic therapy

The application of emerging nanocatalytic drugs in tumor therapy mainly depends on reactive oxygen species (ROS) production in tumors. However, current nanocatalysts have drawbacks such as catalytic inefficiency and high toxicity. Herein, we designed a paradigm of synergy and “division of labor” bimetallic dual active sites single-atom catalyst (SAC) combined with ROS circulation and parallel catalytic therapy for efficient tumor therapy. Both Fe and Co atoms were isolated in the N-doped carbon material with a sharp-angled dodecahedron in a monodisperse state to form an independent bimetallic non-alloy structured atom pair (FeCo-DIA/NC). FeCo-DIA/NC synergistically initiates both Fenton and Fenton-like reactions, directly and simultaneously catalyzing H2O2 and O2 into ROS, including hydroxyl radicals (•OH), superoxide ions (O2•−), and singlet oxygen (1O2). The above ROS are further converted into H2O2 in the acid tumor microenvironment (TME), forming a “ROS Cycle” for highly efficient tumor inhibition. FeCo-DIA/NC exhibits higher activity for Fenton and Fenton-like reactions than single-atom Fe dispersed on N-doped carbon material (Fe-SIA/NC) and single-atom Co dispersed on N-doped carbon material (Co-SIA/NC). The in vitro and in vivo results show that the FeCo-DIA/NC catalyst significantly induces cell apoptosis and inhibits tumor growth. Furthermore, extremely low metal concentrations and high therapeutic effects were achieved simultaneously in FeCo-DIA/NC, highlighting the promising clinical potential. This bimetallic dual-active site monodisperse catalyst provides an important example for applying SACs in the biomedical field, moreover, the highly effective and parallel catalytic “ROS Cycle” provides an opportunity to design nanocatalysts for oncology.

Mesoporous ferrihydrite-supported Pd nanoparticles for enhanced catalytic dehalogenation of chlorinated environmental pollutant

Organic chlorides are a group of ubiquitous environmental pollutants that have attracted wide attention because of their carcinogenetic effect on human. Catalytic hydrodechlorination represents one of the most promising methods for the removal of these contaminants, but it suffers from drawbacks such as catalytic inefficiency and/or instability, and the danger of using H2 as hydrogen source. The relationship between the catalyst structure and its dehalogenation activity has not been completely understood. By combining the advantages of Pd nanocatalyst and mesoporous ferrihydrite (Fh) with its distinctive structure, here we present a new composite material with Pd nanoparticles (NPs) supported onto the Fh (Pd/Fh), which has excellent catalytic dehalogenation performance with a rapid, complete dechlorination of chlorophenol (turnover frequency 25.2 min−1) and the ability to perform well over a wide range of pH and temperature. The superior catalytic property of Pd/Fh can be attributed to the three unique functions of Fh, including: 1) having abundant hydroxyl groups that provide interaction sites with metals for incorporating highly dispersed small Pd NPs; 2) facilitating the fast adsorption of chlorophenol onto the catalyst surface via hydrogen bonding and importantly, 3) working as an electron mediator to greatly enhance the electron transfer from iron or chemicals (e.g., NaBH4) to the catalyst, thereby achieving a synergistic effect between Pd catalyst and support, and an enhanced dechlorination activity. In essence, this work presents a promising catalyst for the efficient dehalogenation of chlorinated environmental pollutants and provides an insight into the relationship between catalyst structure and dehalogenation activity.

Bundle sheath suberisation is required for C4 photosynthesis in a Setaria viridis mutant

C4 photosynthesis provides an effective solution for overcoming the catalytic inefficiency of Rubisco. The pathway is characterised by a biochemical CO2 concentrating mechanism that operates across mesophyll and bundle sheath (BS) cells and relies on a gas tight BS compartment. A screen of a mutant population of Setaria viridis, an NADP-malic enzyme type C4 monocot, generated using N-nitroso-N-methylurea identified a mutant with an amino acid change in the gene coding region of the ABCG transporter, a step in the suberin synthesis pathway. Here, Nile red staining, TEM, and GC/MS confirmed the alteration in suberin deposition in the BS cell wall of the mutant. We show that this has disrupted the suberin lamellae of BS cell wall and increased BS conductance to CO2 diffusion more than two-fold in the mutant. Consequently, BS CO2 partial pressure is reduced and CO2 assimilation was impaired in the mutant. Our findings provide experimental evidence that a functional suberin lamellae is an essential anatomical feature for efficient C4 photosynthesis in NADP-ME plants like S. viridis and have implications for engineering strategies to ensure future food security.

A P3A-Type ATPase and an R2R3-MYB Transcription Factor Are Involved in Vacuolar Acidification and Flower Coloration in Soybean.

The determination of flower color mainly depends on the anthocyanin biosynthesis pathway and vacuolar pH; however, unlike the former, the mechanism of vacuolar acidification in soybean remains uncharacterized at the molecular level. To investigate this mechanism, we isolated four recessive purple–blue EMS-induced flower mutants from the purple flower soybean cultivar, Pungsannamul. The petals of all the mutants had increased pH compared with those of wild Pungsannamul. One of the mutants had a single nucleotide substitution in GmPH4, a regulator gene encoding an MYB transcription factor, and the substitution resulted in a premature stop codon in its first exon. The other three mutants had nucleotide substitutions in GmPH5, a single new gene that we identified by physical mapping. It corresponds to Glyma.03G262600 in chromosome 3 and encodes a proton pump that belongs to the P3A-ATPase family. The substitutions resulted in a premature stop codon, which may be a defect in the ATP-binding capacity of GmPH5 and possibly a catalytic inefficiency of GmPH5. The result is consistent with their genetic recessiveness as well as the high pH of mutant petals, suggesting that GmPH5 is directly involved in vacuolar acidification. We also found that the expression of GmPH5 and several putative “acidifying” genes in the gmph4 mutant was remarkably reduced, indicating that GmPH4 may regulate the genes involved in determining the vacuolar pH of soybean petals.

Development of a Platform for Near-Infrared Photoredox Catalysis.

Over the past decade, chemists have embraced visible-light photoredox catalysis due to its remarkable ability to activate small molecules. Broadly, these methods employ metal complexes or organic dyes to convert visible light into chemical energy. Unfortunately, the excitation of widely utilized Ru and Ir chromophores is energetically wasteful as ∼25% of light energy is lost thermally before being quenched productively. Hence, photoredox methodologies require high-energy, intense light to accommodate said catalytic inefficiency. Herein, we report photocatalysts which cleanly convert near-infrared (NIR) and deep red (DR) light into chemical energy with minimal energetic waste. We leverage the strong spin–orbit coupling (SOC) of Os(II) photosensitizers to directly access the excited triplet state (T1) with NIR or DR irradiation from the ground state singlet (S0). Through strategic catalyst design, we access a wide range of photoredox, photopolymerization, and metallaphotoredox reactions which usually require 15–50% higher excitation energy. Finally, we demonstrate superior light penetration and scalability of NIR photoredox catalysis through a mole-scale arene trifluoromethylation in a batch reactor.

Conformational changes and catalytic inefficiency associated with Mot1-mediated TBP-DNA dissociation.

The TATA-box Binding Protein (TBP) plays a central role in regulating gene expression and is the first step in the process of pre-initiation complex (PIC) formation on promoter DNA. The lifetime of TBP at the promoter site is controlled by several cofactors including the Modifier of transcription 1 (Mot1), an essential TBP-associated ATPase. Based on ensemble measurements, Mot1 can use adenosine triphosphate (ATP) hydrolysis to displace TBP from DNA and various models for how this activity is coupled to transcriptional regulation have been proposed. However, the underlying molecular mechanism of Mot1 action is not well understood. In this work, the interaction of Mot1 with the DNA/TBP complex was investigated by single-pair Förster resonance energy transfer (spFRET). Upon Mot1 binding to the DNA/TBP complex, a transition in the DNA/TBP conformation was observed. Hydrolysis of ATP by Mot1 led to a conformational change but was not sufficient to efficiently disrupt the complex. SpFRET measurements of dual-labeled DNA suggest that Mot1’s ATPase activity primes incorrectly oriented TBP for dissociation from DNA and additional Mot1 in solution is necessary for TBP unbinding. These findings provide a framework for understanding how the efficiency of Mot1’s catalytic activity is tuned to establish a dynamic pool of TBP without interfering with stable and functional TBP-containing complexes.

Alcohol Metabolic Inefficiency: Structural Characterization of Polymorphism-Induced ALDH2 Dysfunctionality and Allosteric Site Identification for Design of Potential Wildtype Reactivators.

Liver mitochondrial aldehyde dehydrogenase 2 (ALDH2) enzyme is responsible for the rapid conversion of acetaldehyde to acetic acid. ALDH2 (E487K) polymorphism results in an inactive allele (ALDH2*2) which cause dysfunctional acetaldehyde metabolism. The 3D structure of an enzyme is crucial to its functionality and a disruption in its structural integrity could result in its metabolic inefficiency and dysfunctionality. Allosteric targeting of polymorphs could facilitate the restoration of wildtype functionalities in ALDH2 polymorphs and serve as an advancement in the treatment of associated diseases. Therefore, structural insights into ALDH2*2 polymorph could reveal the varying degree of alterations which occur at its critical domains and accounts for enzymatic dysfunctionality. In this study, we report the structural characterization of ALDH2*2 polymorph and its critical domains using computational tools. Our findings revealed that the polymorph exhibited significant alterations in stability and flexibility at the catalytic and co-enzyme-binding domain. Moreover, there was an increase in the solvent-exposed surface residues and this indicates structural perturbations. Analysis of the interaction network at ALDH2*2 catalytic domain revealed residual displacement and interaction loss when compared to the wildtype thereby providing insight into the catalytic inefficiency of the polymorph. Interestingly, perturbations induced by ALDH2 polymorphism involves the re-orientation of surface residues, which resulted in the formation of surface exposed pockets. These identified pockets could be potential sites for allosteric targeting. The findings from this study will aid the design of novel site-specific small molecule reactivators with the propensity of restoring wildtype activities for treatment of polymorphic ALDH2 related diseases.

ChemInform Abstract: Direct Enantioselective Aldol Reactions Catalyzed by Calix[4]arene‐Based L‐Proline Derivatives in the Water.

Abstract Two novel p-tert-butylcalix[4]arene-based chiral organocatalysts derived from l -proline have been developed to catalyze direct aldol reactions between cyclohexanone and aromatic aldehydes in water. Under the optimal conditions, high yields (up to 95%), enantioselectivities (up to 90%), and moderate diastereoselectivities (up to 65:35) were obtained. Considering the catalytic inefficiency of sole proline for the aldol reaction in water, these results clearly display the enormous effect of the hydrophobic part of calix[4]arene of compound A.

Perturbing the general base residue Glu166 in the active site of class A β-lactamase leads to enhanced carbapenem binding and acylation.

Most class A β-lactamases cannot hydrolyze carbapenem antibiotics effectively. The molecular mechanism of this catalytic inefficiency has been attributed to the unique stereochemistry of carbapenems, including their 6-α-hydroxyethyl side chain and the transition between two tautomeric states when bound at the active site. Previous studies have shown that the 6-α-hydroxyethyl side chain of carbapenems can interfere with catalysis by forming hydrogen bonds with the deacylation water molecule to reduce its nucleophilicity. Here our studies of a class A noncarbapenemase PenP demonstrate that substituting the general base residue Glu166 with Ser or other residues leads to a significant enhancement of the acylation kinetics by ∼100-500 times toward carbapenems like meropenem. The structures of PenP and Glu166Ser both in apo form and in complex with meropenem reveal that Glu166 is critical for the formation of a hydrogen bonding network within the active site that locks Asn170 in an orientation to impose steric clash with the 6-α-hydroxyethyl side chain of meropenem. The Glu166Ser substitution weakens this network and enables Asn170 to adopt an alternative conformation to avoid steric clash and accommodate faster acylation kinetics. Furthermore, the weakened hydrogen bonding network caused by the Glu166Ser substitution allows the 6-α-hydroxyethyl moiety to adopt a catalytically favorable orientation as seen in class A carbapenemases. In summary, our data identify a previously unreported role of the universally conserved general base residue Glu166 in impeding the proper binding of carbapenems by restricting their 6-α-hydroxyethyl group.

Direct enantioselective aldol reactions catalyzed by calix[4]arene-based l-proline derivatives in the water

Two novel p-tert-butylcalix[4]arene-based chiral organocatalysts derived from l-proline have been developed to catalyze direct aldol reactions between cyclohexanone and aromatic aldehydes in water. Under the optimal conditions, high yields (up to 95%), enantioselectivities (up to 90%), and moderate diastereoselectivities (up to 65:35) were obtained. Considering the catalytic inefficiency of sole proline for the aldol reaction in water, these results clearly display the enormous effect of the hydrophobic part of calix[4]arene of compound A.

The enigmatic thymine DNA glycosylase

When it was first isolated from extracts of HeLa cells in Josef Jiricny's laboratory, the thymine DNA glycosylase (TDG) attracted attention because of its ability to remove thymine, i.e. a normal DNA base, from G·T mispairs. This implicated a function of DNA base excision repair in the restoration of G·C base pairs following the deamination of a 5-methylcytosine. TDG turned out to be the founding member of a newly emerging family of mismatch-directed uracil-DNA glycosylases, the MUG proteins, that act on a comparably broad spectrum of base lesion including G·U as the common, most efficiently processed substrate. However, because of its apparent catalytic inefficiency, some have considered TDG a poor DNA repair enzyme without an important biological function. Others have reported 5-meC DNA glycosylase activity to be associated with TDG, thrusting the enzyme into limelight as a possible DNA demethylase. Yet others have found the glycosylase to interact with transcription factors, implicating a function in gene regulation, which appears to be critically important in developmental processes. This article reviews all these developments in view of possible biological functions of this multifaceted DNA glycosylase.

GTPase Catalysis by Ras and Other G-proteins: Insights from Substrate Directed SuperImposition

Comparisons of different protein structures are commonly carried out by superimposing the coordinates of the protein backbones or selected parts of the proteins. When the objective is analysis of similarities and differences in the enzyme's active site, there is an inherent problem in using the same domains for the superimposition. In this work we use a comparative approach termed here “Substrate Directed SuperImposition” (SDSI). It entails the superimposition of multiple protein–substrate structures using exclusively the coordinates of the comparable substrates. SDSI has the advantage of unbiased comparison of the active-site environment from the substrate's point of view. Our analysis extends previous usage of similar approaches to comparison of enzyme catalytic machineries.We applied SDSI to various G-protein structures for dissecting the mechanism of the GTPase reaction that controls the signaling activity of this important family. SDSI indicates that dissimilar G-proteins stabilize the transition state of the GTPase reaction similarly and supports the commonality of the critical step in this reaction, the reorientation of the critical arginine and glutamine. Additionally, we ascribe the catalytic inefficiency of the small G-protein Ras to the great flexibility of its active site and downplay the possible catalytic roles of the Lys16 residue in Ras GTPase. SDSI demonstrated that in contrast to all other Gly12 Ras mutants, which are oncogenic, the Gly12→Pro mutant does not interfere with the catalytic orientation of the critical glutamine. This suggests why this mutant has a higher rate of GTP hydrolysis and is non-transforming. Remarkably, SDSI also revealed similarities in the divergent catalytic machineries of G-proteins and UMP/CMP kinase. Taken together, our results promote the use of SDSI to compare the catalytic machineries of both similar and different classes of enzymes.

Engineered Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Binds theAntiConformation of NAD+Nicotinamide but Does Not Experience A-Specific Hydride Transfer

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-dependent oxidoreductase which catalyzes the oxidative phosphorylation ofd-glyceraldehyde-3-phosphate (G3P) to form 1,3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. GAPDH is classified as a B-specific oxidoreductase. The inspection of several crystal structures of GAPDHs indicates that the efficient hydride transfer from the hemithioacetal intermediate to the C4 position of the pyridiniumsiface requires optimal nicotinamidium–protein contacts for a suitable pyridinium-ring orientation. In previous studies carried out onEscherichia coliGAPDH (C. Corbier, A. Mougin, Y. Mely, H. W. Adolph, M. Zeppezauer, D. Gerard, A. Wonacott, and G. Branlant,Biochimie72, 545–554, 1990; J. Eyschen, C. Corbier, B. Vitoux, G. Branlant, and M. T. Cung,Protein Pept. Lett.1, 19–24, 1994), the role of the invariant Asn 313 residue, as an anchor which favors thesynorientation of the nicotinamide ring, was examined. Here, we report further investigations on the molecular factors responsible for the cofactor stereospecificity. Two single [Gly317] and [Ala317] GAPDH mutants and one double [Thr313-Gly317] GAPDH mutant were constructed on the basis of a molecular modelling study from the crystal structure of holo GAPDH fromE. coli(E. Duée, L. Olivier-Deyris, E. Fanchon, C. Corbier, G. Branlant, and O. Dideberg,J. Mol. Biol.257, 814–838, 1996). TheKdconstants of [Ala317], [Gly317], and [Thr313-Gly317] GAPDH mutants for NAD are 5, 13, and 300 times higher than that of wild-type GAPDH. Transferred nuclear Overhauser effect spectroscopy demonstrates that the wild-typesynorientation of bound nicotinamide remains unchanged in the [Gly317] and [Ala317] mutants, whereas a conformational equilibrium between thesynandantiforms occurs in the [Thr313-Gly317] double mutant with a preference for theanticonformer. Although the double mutant preferably binds the nicotinamide ring in ananticonformation, it still exhibits B hydride transfer stereospecificity. Yet, the catalytic efficiency is much less than that of the wild type. This indicates that the holo GAPDH mutant fraction with anantiorientation of bound NAD is not capable of forming the ternary complex with G3P which would be required for an efficient A-specific catalytic process. The reasons of this catalytic inefficiency are discussed in relation with the historical and functional models which were advanced to explain the stereospecificity of NAD(P)-dependent dehydrogenases.

Structural features that determine the enzymatic potency and specificity of human angiogenin: threonine-80 and residues 58-70 and 116-123.

Human angiogenin (Ang), a homologue of bovine pancreatic ribonuclease A (RNase A), is a potent inducer of blood vessel formation. It exerts a ribonucleolytic activity that is 10(5)-10(6)-fold lower than that of RNase A but nonetheless essential for biological action. Previous studies revealed some of the structural features of Ang that underlie its catalytic inefficiency: Gln-117 blocks the space corresponding to the pyrimidine binding site of RNase A and Ang lacks the disulfide loop 65-72 that forms most of the purine binding site of RNase A. Additional features have now been identified by mutagenesis and kinetics. Thr-80, which hydrogen-bonds to the pyrimidine-binding residue Thr-44, plays an important part in attenuating activity and in determining pyrimidine specificity: mutation to Ala increases activity toward cytidylyl substrates by 11-15-fold but has only a minimal effect on cleavage of uridylyl substrates. The properties of T44A/T80A and Q117A/T80A double mutants demonstrate that these changes are mediated by Thr-44 and are largely independent of the blockage by Gln-117. The side chain of Ser-118 also suppresses enzymatic activity: S118A is 5-7-fold more effective than Ang. This increase appears to reflect the loss of a hydrogen bond with Asp-116 that helps to orient Gln-117. The effects of deleting residues 119-123 suggest that main-chain atoms of the C-terminal 3(10) helix make a small further contribution. Finally, the significance of the absence of the RNase A loop 65-72 from Ang has been investigated by reexamining the earlier derivative ARH-I (in which Ang residues 58-70 have been replaced by residues 59-73 of RNase) and generating new derivatives of this hybrid protein. The results suggest that the RNase A segment of ARH-I not only provides more effective purine recognition but also counteracts the deleterious effects of Gln-117 and Thr-80 on the pyrimidine site.

Product inhibition during ion: Solute cotransport is an alternative to leaks as a cause of low accumulations

Ion: solute cotransporters frequency are incapable of achieving equilibrium between the solute accumulation and the transmembrane difference of the electrochemical potential of the ion. The presence of uncoupled flows of ion and solutes (leaks) is often advanced as an explanation. Here an alternative is discussed. The net accumulation of solute may be so slow that equilibrium can never be attained at finite times (e.g., several hours). Cotransporters may exhibit strong product inhibition, and the net influx of solute approaches zero far from equilibrium. The inherent slowness of net transport under these conditions is termed catalytic inefficiency. The likelihood that galactoside: H+ cotransport inEscherichia coli, hexose: H+ cotransport inChlorella vulgaris, andd-glucose: Na+ cotransport in brush-border membranes exhibit catalytic inefficiency is examined. The existence of strong product inhibition complicates the determination of the stoichiometry of cotransport and the characterization of chemically modified or mutant cotransporters.

Die H 2S-oxidation an aktiver kohle—ein elektrochemischer prozess?

Proceeding from the observation that a galvanic cell can be formed with activated carbon as electrode material and aqueous solutions of H 2S and oxygen, a mechanism is submitted for discussion, according to which the oxidation of H 2S catalyzed by activated carbon takes place by this very formation of cells on the microscopic surfaces of the carbon. The known effects of H 2O, H + ions, as well as Fe and I content of the activated carbon on catalysis may be explained by this model. The catalytic inefficiency of dry activated carbon with respect to the oxidation of H 2S with oxygen-containing gases at ambient temperature is due to the fact that electrolyte is indispensable for electrochemical processes. Carbon hexagon layers with reversibly chemisorbed O 2 behave as oxygen electrode while layers with adsorbed H 2S act as fuel electrode. Only after addition of electrolyte elemental sulphur can separate on the fuel electrode. The pH value can drop far below pH 2 due to H 2SO 4 formed in a side reaction. With such acid concentrations, H 3S + ions are increasingly formed. Since H 3S + is no longer accessible to electrochemical oxidation, catalysis is retarded within these pH ranges. Iron bonded in complex form to the layers behaves as an electron acceptor. By this, the layers and the adsorbed H 2S are positively polarized which allows oxidation of the H 2S to sulphuric acid. The negatively polarized iron assumes the function of the oxygen electrode. Iodine blocks the direct access of H 2S to the layer or fuel electrode respectively. By direct chemical reaction, also within the strongly acidic range, elemental iodine reacts with H 2S forming elemental sulphur. The iodide ions formed are subject to electrochemical oxidation to I 2 on the carbon layer. The special advantage of the electrochemical oxidation mechanism is that the O 2 and H 2S molecules must not necessarily be in direct contact.

An inherited molecular lesion of erythrocyte pyruvate kinase. Identification of a kinetically aberrant isozyme associated with premature hemolysis.

Atypical cases of heritable hemolytic anemia have been noted that conform clinically and biochemically to anemias of the pyruvatekinase (PK)-deficient type, except for the presence of apparently adequate quantities of erythrocyte-PK activity by the usual assay procedure. Investigations of four such anomalous cases, occurring in two unrelated families, are presented. Erythrocytes contained a kinetically aberrant isozyme of pyruvate kinase (PK(2)). Michaelis constants for the pathologic isozyme relative to phosphoenolpyruvate were over 10-fold greater than control values, but no kinetic abnormality was evident for the second substrate, adenosine diphosphate. PK(2) exhibited a pH optimum almost 1 U lower than the wild enzyme form (PK(1)). Significant differences were also evident in the functional stabilities of the isozymes. Leukocytes were unaffected. Family studies revealed paternal heterozygosity for quantitative PK deficiency of the usual type. Clinically normal maternal relatives and some siblings demonstrated intermediate deviations in erythrocyte-PK kinetics and reaction characteristics compatible with coexistence of normal PK(1) and kinetically abnormal PK(2). Hemolytic anemia in the propositi appeared to require simultaneous inheritance of the gene governing PK(2) production and its presumed allele resulting in quantitative PK deficiency. Both genetic defects were traced through three generations, the defective gene in both instances apparently resident on autosomes.A revision of the PK assay technique is suggested, since catalytic inefficiency of PK(2) was manifested only at low substrate concentrations and was therefore undetectable at the relatively high phosphoenolpyruvate levels employed in the conventional assay.