Transfer of pro-R hydrogen from NADH to dihydroxyacetonephosphate by sn-glycerol-1-phosphate dehydrogenase from the archaeon Methanothermobacter thermautotrophicus

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of the sn-glycerol-1-phosphate backbone of archaeal lipids. [4-3H]NADH that had 3H at the R side was produced from [4-3H]NAD and glucose with glucose dehydrogenase (a pro-S type enzyme). The 3H of this [4-3H]NADH was transferred to dihydroxyacetonephosphate during the sn-glycerol-1-phosphate dehydrogenase reaction. On the contrary, in a similar reaction using alcohol dehydrogenase (a pro-R type enzyme), 3H was not incorporated into glycerophosphate. These results confirmed a prediction of the tertiary structure of sn-glycerol-1-phosphate dehydrogenase by homology modeling.     

Kinetic properties of a sn-glycerol-3-phosphate dehydrogenase purified from the unicellular alga Chlamydomonas reinhardtii

A sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) has been purified from the unicellular green alga Chlamydomonas reinhardtii 3400-fold to a specific activity of 34 mumol/mg protein per min by a simple procedure involving two chromatographic steps on affinity dyes. The pH optimum for reduction of dihydroxyacetone phosphate was 6.8 and for glycerol 3-phosphate oxidation it was 9.5. In the direction of dihydroxyacetone phosphate reduction, the enzyme showed Michaelis-Menten kinetics. The enzyme reacted specifically with NADH and dihydroxyacetone phosphate as substrates with affinity constants of 16 and 12 microM, respectively. Product inhibition as well as competitive inhibition pattern indicated a random-bi-bi reaction mechanism for sn-glycerol-3-phosphate dehydrogenase from C. reinhardtii. The effective control of dihydroxyacetone reduction catalysed via this enzyme by ATP, Pi and NAD gave evidence for a physiological role of the enzyme in plastidic glycolysis.     

Active site of Zn(2+)-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261) is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A) of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.     

Identification of sn-glycerol-1-phosphate dehydrogenase activity from genomic information on a hyperthermophilic archaeon, Sulfolobus tokodaii strain 7

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme.     

The crystal structure of (S)-3-O-geranylgeranylglyceryl phosphate synthase reveals an ancient fold for an ancient enzyme

We report crystal structures of the citrate and sn-glycerol-1-phosphate (G1P) complexes of (S)-3-O-geranylgeranylglyceryl phosphate synthase from Archaeoglobus fulgidus (AfGGGPS) at 1.55 and 2.0 A resolution, respectively. AfGGGPS is an enzyme that performs the committed step in archaeal lipid biosynthesis, and it presents the first triose phosphate isomerase (TIM)-barrel structure with a prenyltransferase function. Our studies provide insight into the catalytic mechanism of AfGGGPS and demonstrate how it selects for the sn-G1P isomer. The replacement of "Helix 3" by a "strand" in AfGGGPS, a novel modification to the canonical TIM-barrel fold, suggests a model of enzyme adaptation that involves a "greasy slide" and a "swinging door." We propose functions for the homologous PcrB proteins, which are conserved in a subset of pathogenic bacteria, as either prenyltransferases or being involved in lipoteichoic acid biosynthesis. Sequence and structural comparisons lead us to postulate an early evolutionary history for AfGGGPS, which may highlight its role in the emergence of Archaea.     

sn-Glycerol-3-phosphate dehydrogenase and its interaction with nitrate reductase in wild-type and hem mutant strains of Staphylococcus aureus

Staphylococcus aureus has membrane-associated sn-glycerol-3-phosphate dehydrogenase activity that is strongly activated by detergents. The enzyme can be measured spectrophotometrically in intact cells in assay systems containing lauryldimethylamine oxide (Ammonyx LO). The dehydrogenase activity was located exclusively in the membrane fraction of cells grown with glycerol under aerobic conditions or under anaerobic conditions with the addition of nitrate; there was no evidence of multiple forms. Development of sn-glycerol-3-phosphate dehydrogenase activity was studied with suspensions of cells grown previously under semianaerobic conditions with glucose and nitrate. The wild-type strain rapidly formed the enzyme when incubated with glycerol under aerobic conditions or under semianaerobic conditions in the presence of nitrate. Under similar conditions, suspensions of hem mutant H-14 required the addition of hemin. Induction of the enzyme was strongly repressed by glucose with both organisms. A procedure was established to obtain cells of mutant H-14 with sn-glycerol-3-phosphate dehydrogenase and nitrate reductase activities, but which could not link the systems unless supplemented with hemin. The coupled activity could also be reconstructed in vitro by the addition of hemin to the depleted membranes.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.     

Biosynthesis of ether-type polar lipids in archaea and evolutionary considerations.

This review deals with the in vitro biosynthesis of the characteristics of polar lipids in archaea along with preceding in vivo studies. Isoprenoid chains are synthesized through the classical mevalonate pathway, as in eucarya, with minor modifications in some archaeal species. Most enzymes involved in the pathway have been identified enzymatically and/or genomically. Three of the relevant enzymes are found in enzyme families different from the known enzymes. The order of reactions in the phospholipid synthesis pathway (glycerophosphate backbone formation, linking of glycerophosphate with two radyl chains, activation by CDP, and attachment of common polar head groups) is analogous to that of bacteria. sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of the sn-glycerol-1-phosphate backbone of phospholipids in all archaea. After the formation of two ether bonds, CDP-archaeol acts as a common precursor of various archaeal phospholipid syntheses. Various phospholipid-synthesizing enzymes from archaea and bacteria belong to the same large CDP-alcohol phosphatidyltransferase family. In short, the first halves of the phospholipid synthesis pathways play a role in synthesis of the characteristic structures of archaeal and bacterial phospholipids, respectively. In the second halves of the pathways, the polar head group-attaching reactions and enzymes are homologous in both domains. These are regarded as revealing the hybrid nature of phospholipid biosynthesis. Precells proposed by W?chtersh?user are differentiated into archaea and bacteria by spontaneous segregation of enantiomeric phospholipid membranes (with sn-glycerol-1-phosphate and sn-glycerol-3-phosphate backbones) and the fusion and fission of precells. Considering the nature of the phospholipid synthesis pathways, we here propose that common phospholipid polar head groups were present in precells before the differentiation into archaea and bacteria.     

Glycerol catabolism in Aspergillus nidulans

Glycerol is catabolized in Aspergillus nidulans by glycerol kinase and a mitochondrial FAD-dependent sn-glycerol 3-phosphate dehydrogenase. The levels of both enzymes are controlled by carbon catabolite repression and by specific induction. Biochemical and genetical analyses show that dihydroxyacetone and D-glyceraldehyde are converted into glycerol and then catabolized by the same pathway. D-Glyceraldehyde can be reduced by NADP(+)-dependent glycerol dehydrogenase or by alcohol dehydrogenase I, while dihydroxyacetone is only reduced by the first enzyme. Three new glycerol non-utilizing mutants have been found. These three mutations define three hitherto unknown loci, glcE, glcF and glcG. The mutation in glcG leads to a greatly decreased sn-glycerol-3-phosphate dehydrogenase activity.     

Glycerol 3-phosphate analogues as metabolic inhibitors in Escherichia coli, 3-hydroxy-4-oxobutyl-1-phosphonate, a drug that interferes with normal phosphoglyceride metabolism.

3-Hydroxy-4-oxobutyl-1-phosphonate, the phoshonic acid analogue of glyceraldehyde 3-phosphate, enters Escherichia coli via the glycerol 3-phosphate transport system. There is no differential effect upon the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although the accumulation of proteins was less effected. Examination of the phospholipids revealed that phosphatidylglycerol accumulation was most severely inhibited and cardiolipin accumulation was least affected. Concentrations of glyceraldehyde 3-phosphate and its phosphonic acid analogue that markedly inhibit macromolecular and phosphoglyceride biosynthesis have no effect upon the intracellular nucleoside triphosphate pool size. The phosphonate is a competitive inhibitor of sn-glycerol 3-phosphate in reactions catalyzed by acyl coenzyme A:sn-glycerol-3-phosphate acyltransferase and CDP-diacylglycerol:sn-glycerol-3-phosphate phosphatidyltransferase. A Km mutant for the former enzyme was susceptible to the phosphansferase activity. Studies with mutant strains ruled out the aerobic glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate synthase, and fructose-1,6-biphosphate aldolase as the primary sites of action.     

Regulation of malic enzyme and mitochondrial alpha-glycerophosphate dehydrogenase by thyroid hormones, insulin, and glucocorticoids in cultured hepatocytes

Hepatocytes isolated from normal adult rats were cultured under serum-free conditions. Induction of mitochondrial alpha-glycerophosphate dehydrogenase (glycerol 3-phosphate dehydrogenase) (EC 1.1.99.5; sn-glycerol-3-phosphate: (acceptor) oxidoreductase) and cytosolic malic enzyme (EC 1.1.1.40; L-malate-NADP+ oxidoreductase (decarboxylating)) by 3,3'-5-triiodo-L-thyronine (triiodothyronine) in the culture medium follows the same time course as the in vivo response to thyroid hormones. The addition of 1 microM cycloheximide blocks the triiodothyronine response. Thyroxine is also capable of stimulating the activities of both enzymes. Although increases in alpha-glycerophosphate dehydrogenase and malic enzyme activities are observed when triiodothyronine is added to the culture medium for 3 days (62% and 36%, respectively), in the presence of insulin and cortisol the response is significantly greater. Dexamethasone is more potent than cortisol in increasing triiodothyronine action. In the presence of bovine serum albumin, to prevent metabolism of triiodothyronine, hepatocytes show increased enzyme activity at concentrations as low as 10(-10) M triiodothyronine.     

Did Archaeal and Bacterial Cells Arise Independently from Noncellular Precursors? A Hypothesis Stating That the Advent of Membrane Phospholipid with Enantiomeric Glycerophosphate Backbones Caused the Separation of the Two Lines of Descent

One of the most remarkable biochemical differences between the members of two domains Archaea and Bacteria is the stereochemistry of the glycerophosphate backbone of phospholipids, which are exclusively opposite. The enzyme responsible to the formation of Archaea-specific glycerophosphate was found to be NAD(P)-linked sn-glycerol-1-phosphate (G-1-P) dehydrogenase and it was first purified from Methanobacterium thermoautotrophicum cells and its gene was cloned. This structure gene named egsA (enantiomeric glycerophosphate synthase) consisted of 1,041 bp and coded the enzyme with 347 amino acid residues. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity except for NAD-binding region with that of NAD(P)-linked sn-glycerol-3-phosphate (G-3-P) dehydrogenase of Escherichia coli which catalyzes the formation of G-3-P backbone of bacterial phospholipids, while the deduced protein sequence of the enzyme revealed some similarity with bacterial glycerol dehydrogenases. Because G-1-P dehydrogenase and G-3-P dehydrogenase would originate from different ancestor enzymes and it would be almost impossible to interchange stereospecificity of the enzymes, it seems likely that the stereostructure of membrane phospholipids of a cell must be maintained from the time of birth of the first cell. We propose here the hypothesis that Archaea and Bacteria were differentiated by the occurrence of cells enclosed by membranes of phospholipids with G-1-P and G-3-P as a backbone, respectively. Received: 24 March 1997 / Accepted: 21 May 1997     

Ancestral lipid biosynthesis and early membrane evolution

Archaea possess unique membrane phospholipids that generally comprise isoprenoid ethers built on sn-glycerol-1-phosphate (G1P). By contrast, bacterial and eukaryal membrane phospholipids are fatty acid esters linked to sn-glycerol-3-phosphate (G3P). The two key dehydrogenase enzymes that produce G1P and G3P, G1PDH and G3PDH, respectively, are not homologous. Various models propose that these enzymes originated during the speciation of the two prokaryotic domains, and the nature (and even the very existence) of lipid membranes in the last universal common ancestor (cenancestor) is subject to debate. G1PDH and G3PDH belong to two separate superfamilies that are universally distributed, suggesting that members of both superfamilies existed in the cenancestor. Furthermore, archaea possess homologues to known bacterial genes involved in fatty acid metabolism and synthesize fatty acid phospholipids. The cenancestor seems likely to have been endowed with membrane lipids whose synthesis was enzymatic but probably non-stereospecific.     

Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase

One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn²⁺ cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn²⁺ cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH.     

Characterization of active and latent forms of the membrane-associated sn-glycerol-3-phosphate acyltransferase of Escherichia coli

The intrinsically active, sn-glycerol-3-phosphate acyltransferase present in membranes prepared from both wild type Escherichia coli and from strains which overproduce the enzyme can be kinetically distinguished from a latent enzyme species which is unmasked by solubilization and reconstitution. Both membrane-associated and solubilized/reconstituted enzyme preparations exhibited cooperativity with respect to sn-glycerol-3-phosphate and palmitoyl-coenzyme A substrates; positive cooperativity in membranes toward palmitoyl-coenzyme A (napp = 4) and negative cooperativity toward sn-glycerol-3-phosphate (napp = 0.75) were significantly altered upon solubilization and reconstitution. Since the degree of alteration increased with the amount of sn-glycerol-3-P acyltransferase present in the membranes, a detergent-dissociable homooligomerization of the sn-glycerol-3-phosphate acyltransferase was considered as an underlying mechanism. This possibility was investigated by changing the protein-to-Triton X-100 ratio of homogeneous enzyme prior to reconstitution and then analyzing the subsequent migration of samples on a Sephacryl S-300 sizing column. The elution positions were consistent with monomeric and dimeric polypeptide bound to micelles of Triton X-100. Hill coefficients for monomeric, reconstituted enzyme preparations were comparable to those obtained for the active, membrane-associated sn-glycerol-3-phosphate acyltransferase. The reduced cooperativity of dimeric, reconstituted enzyme preparations correlated closely to the Hill coefficient values obtained for latent, solubilized/reconstituted sn-glycerol-3-phosphate acyltransferase from membranes of Escherichia coli which overproduce the enzyme. The physiological significance of these findings is discussed.     

Intraorganelle localization and substrate specificities of the mitochondrial acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase and acyl-CoA: 1-acyl-sn-glycerol-3-phosphate O-acyltransferase from potato tubers and pea leaves

The mitochondrial sn-glycerol-3-phosphate and 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1-acyl-sn-glycerol-3-phosphate O-acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn-glycerol-3-phosphate O-acyltransferase behaved like a soluble protein of the intermembrane space. The sn-glycerol-3-phosphate O-acyltransferase of both potato and pea mitochondria used sn-glycerol-3-phosphate but not dihydroxyacetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1-acyl-sn-glycerol-3-phosphate which subsequently served as substrate for the second acylation reaction at its C-2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl-CoA than with the corresponding acyl-(acyl carrier protein) thioesters. When different acyl-CoA thioesters were offered separately, the sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl-CoA thioesters. Furthermore, these enzymes preferentially used oleoyl- and linoleoyl-CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups.     

Comparison of glycerophosphate acyltransferases from Euglena chloroplasts and microsomes

The acylation of sn-glycerol 3-phosphate is a common reaction in the pathways leading to the biosynthesis of glycerol-derived phospholipids, galactolipids, and sulfolipids. Enzymes catalyzing this reaction have been solubilized from Euglena chloroplasts, microsomes, and mitochondria (B. A. Boehler and M. L. Ernst-Fonberg (1976) Arch. Biochem. Biophys. 175, 229-235; L. V. Grobovsky, S. Hershenson, and M. L. Ernst-Fonberg (1979) FEBS Lett. 102, 261-264). Some characteristics of the reactions catalyzed by the acyl-CoA:sn-glycerol-3-phosphate O-acyltransferases (EC 2.3.1.15) solubilized from chloroplasts and microsomes of Euglena have been compared. Although the two enzymes have some common features, including stimulation by bovine serum albumin and phosphatidyl choline and sensitivity to sulfhydryl-binding reagents, they differ in their stabilities and responses to salt and glycerol. They exhibit different acyl-CoA substrate dependency curves. The proportions of monoacyl sn-glycerol-3-phosphate acyltransferase activity differ in the two solubilized enzyme preparations, and different products are produced by each of the glycerophosphate acyltransferases solubilized from chloroplasts and microsomes, respectively. Neither glycerophosphate acyltransferase will use palmitoyl- or myristoyl-acyl carrier protein (ACP) as a substrate whereas both use the corresponding CoA esters. Neither is inhibited by ACP, but the enzyme from microsomes is inhibited by CoA.     

Increased activity of glycerol 3-phosphate dehydrogenase and other lipogenic enzymes in human bladder cancer.

Common molecular changes in cancer cells are high carbon flux through the glycolytic pathway and overexpression of fatty acid synthase, a key lipogenic enzyme. Since glycerol 3-phosphate dehydrogenase creates a link between carbohydrates and the lipid metabolism, we have investigated the activity of glycerol 3-phosphate dehydrogenase and various lipogenic enzymes in human bladder cancer. The data presented in this paper indicate that glycerol 3-phosphate dehydrogenase activity in human bladder cancer is significantly higher compared to adjacent non-neoplastic tissue, serving as normal control bladder tissue. Increased glycerol 3-phosphate dehydrogenase activity is accompanied by increased enzyme activity, either directly (fatty acid synthase) or indirectly (through ATP-citrate lyase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and citrate synthase) involved in fatty acid synthesis. Coordinated upregulation of glycerol 3-phosphate dehydrogenase and lipogenic enzymes activities in human bladder cancer suggests that glycerol 3-phosphate dehydrogenase supplies glycerol 3-phosphate for lipid biosynthesis.     

A study of the kinetics and mechanism of rabbit muscle l-glycerol 3-phosphate dehydrogenase

1. The kinetics of oxidation of l-glycerol 3-phosphate by NAD(+) and of reduction of dihydroxyacetone phosphate by NADH catalysed by rabbit muscle glycerol 3-phosphate dehydrogenase were studied over the range pH6-9. 2. The enzyme was found to catalyse the oxidation of glyoxylate by NAD(+) at pH8.0 and the kinetics of this reaction were also studied. 3. The results are consistent with a compulsory mechanism of catalysis for glycerol 3-phosphate oxidation and dihydroxyacetone phosphate reduction in the intermediate regions of pH, but modifications to the basic mechanism are required to fully explain results at the extremes of the pH range, with these substrates and for glyoxylate oxidation at pH8.0.