Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Israel: dissemination of global clones and unique features.

From 2006 to 2009, 315 clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were collected from 5 hospitals across Israel. Most isolates (64%) were related to the global clones spa types t001-SCCmec-I (SCCmec-I stands for staphylococcal cassette chromosome mec type I) (n = 99; 31%), t002-SCCmec-II (n = 82; 26%), and t008-SCCmec-IV (n = 21; 7%), five of which were identified as MRSA strain USA-300. Seventeen strains unique to Israel were identified. SCCmec types IV and V were common among hospital-acquired isolates.

Mucormycosis in a liver allograft: salvage re-transplantation and targeted immunosuppressive management.

Zygomycetes infection is associated with a high mortality in transplant populations. We describe a child with liver allograft Rhizopus oryzae infection who was salvaged by liver re-transplantation. A 10-year-old child presented with anastomotic bile leak that was repaired. A combined antibiotics and voriconazole regimen was introduced for Escherichia coli and Candida krusei growth in the peritoneal fluid. Despite broad antibiotic and antifungal coverage, the patient continued to have an ongoing infection. A follow-up computed tomography scan 8 weeks later showed 2 liver abscesses infiltrating the stomach and the diaphragm, with splenic infarcts and pericardial effusion. Aspirated samples from the liver abscess and the pericardial fluid revealed R. oryzae. Immunosuppression was discontinued and an antifungal regimen combining amphotericin B, posaconazole, and caspofungin was introduced. After 3 weeks of treatment with control of the systemic signs of infection, a positron emission tomography showed the fluorescence stain limited to the liver. With infection confined to the liver, the child underwent liver re-transplantation, splenectomy, and partial gastrectomy. Immunosuppression was reintroduced with recovery of the immune response observed by the CD4 cells adenosine triphophate release (Cylex(™) ImmuKnow(®) assay) and posaconazole was continued for another year. At 3-year follow-up, the child maintained normal graft function. We conclude that discontinuation of immunosuppression combined with a modern antifungal regimen may allow salvage re-transplantation in patients with liver mucormycosis.

Chronic granulomatous disease of childhood: an unusual cause of recurrent uncommon infections in a 61-year-old man.

Chronic granulomatous disease (CGD) is a rare congenital immunodeficiency that affects 1 : 250,000 of the population, which is characterized by recurrent bacterial and fungal infections and by granuloma formation. We investigated a 61-year-old man presented with a 20-year history of a relapsing skin rash appearing as mildly pruritic and erythematous plaques affecting various body regions. Cutaneous biopsies were taken and sent for histology and tissue culture. Leucocyte function was assessed by determining the generation of reactive oxygen species. Bactericidal activity was assessed in the presence of autologous and homologous sera. Western blotting was performed for protein analysis of the reduced nicotinamide adenine dinucleotide phosphate oxidase system, and mutation screening was carried out using PCR amplification and sequence analysis. Examination of biopsies obtained from lesional skin indicated a suppurative granulomatous process. Tissue cultures grew Aspergillus nidulans and Aspergillus fumigatus (confirmed by PCR). A. nidulans has often been associated with CGD, and the leucocyte function tests supported this diagnosis. Direct DNA sequencing led to the identification of a hemizygous missense novel mutation in CYBB (c.907C>T), which predicts a p.His303Tyr amino-acid substitution in gp91-phox, thus confirming the diagnosis of CGD. In conclusion, we report a case of a rare inherited immunodeficiency, CGD, in a 61-year-old man, and describe the novel hemizygous missense mutation underlying the condition. Mild forms of usually fatal immunodeficiencies should be considered when assessing the occurrence of unusual infectious diseases in apparently healthy people.

Radiological findings of early invasive pulmonary aspergillosis in immune-compromised patients.

Data on the radiological features of invasive pulmonary aspergillosis (IPA) in early stages is scanty. Detection of Aspergillus (ASP) species in broncho-alveolar (BAL) fluid by polymerase chain reaction (PCR) enables early diagnosis of IPA. This study describes the radiological features of early stages of IPA. Chest computerized tomography (CT) films of 22 consecutive immune-compromised patients with IPA diagnosed with the aid of ASP PCR testing from BAL fluid were characterized and compared to that of 18 similar patients diagnosed with traditional bacteriological methods and to data from the literature. It was found that patients diagnosed with the aid of ASP PCR testing tended to have focal disease as manifested by more 11-30 mm nodules with halo (68% vs. 33%, p = 0.04), more focal ground glass (single area 32% vs. 6%, p = 0.05, patchy 32% vs. 0%, p = 0.01) and less diffuse ground glass (0% vs. 22%, p = 0.03), less cavitations (5% vs. 28%, p = 0.05) and less consolidations (segmental 14% vs. 50%, p = 0.02 and diffuse 14% vs. 67%, p = 0.001). It was concluded that the radiological appearance of early IPA diagnosed with the aid of PCR testing included mainly discrete small nodules with halo and focal ground glass, representing the early stage of the disease.

Clinical impact of a PCR assay for rapid identification of Klebsiella pneumoniae in blood cultures.

The clinical impact of a rapid PCR identification assay for Klebsiella pneumoniae in positive blood cultures was prospectively evaluated. Multivariate analysis identified the rapid PCR assay as the only significant factor in decreasing the time lapse preceding the initiation of appropriate antimicrobial therapy (hazards ratio, 3.03; confidence interval, 1.62 to 5.68; P, 0.001).

High affinity esterification of eicosanoid precursor fatty acids by platelets.

We have examined the relative rates of uptake of several fatty acids into washed, human platelets by measuring incorporation into cellular phospholipids. In the presence of 15 microM fatty acid-free albumin and with radioactive fatty acid concentrations of 5-500 nM, esterification into phospholipid was linear with time and platelet concentration and saturable with respect to fatty acid concentration. Two distinct classes of uptake rate were observed. Arachidonate and 5,8,11,14,17-eicosapentaenoate exhibited high affinity, relatively rapid incorporation into platelet phospholipids at pH 6.5: apparent Michaelis constant (Km) = 30 nM, apparent maximum velocity (Vmax) = 28 pmol/min per 10(9) platelets. Two other eicosanoid precursors, 5,8,11-eicosatrienoate and 8,11,14-eicosatrienoate, exhibited the same Vmax, but Km of 85 and 60 nM, respectively. Under the same conditions, stearate, oleate, and linoleate were incorporated into phospholipids much less efficiently (Vmax approximately 8 pmol/10(9) cells per min, apparent Km greater than or equal to 170 nM). Qualitatively similar results were found at pH 7.4. Uptake of radiolabeled, rapid-uptake fatty acids was not diminished by the presence of excess, unlabeled, slow-uptake fatty acids. Thus, the specificity of this esterification system resembles that of the arachidonate-specific, long-chain acyl-CoA synthetase present in platelets. It may represent the expression in vivo of the synthetase, although the apparent affinity of the synthetase for fatty acid is much less. This esterification system probably represents the physiologic mechanism for platelet arachidonate uptake, whereby arachidonate is collected from plasma, despite the fact that its concentration is considerably lower than that of other plasma fatty acids.

2,4-Dienoyl-coenzyme A reductase deficiency: a possible new disorder of fatty acid oxidation.

Several inherited disorders of fatty acid beta-oxidation have been described that relate mainly to saturated precursors. This study is the first report of an enzyme defect related only to unsaturated fatty acid oxidation and provides the first in vivo evidence that fat oxidation in humans proceeds by the reductase-dependent pathway. The patient was a black female, presenting in the neonatal period with persistent hypotonia. Biochemical studies revealed hyperlysinemia, hypocarnitinemia, normal organic acid profile, and an unusual acylcarnitine species in both urine and blood. The new metabolite was positively identified by mass spectrometry as 2-trans,4-cis-decadienoylcarnitine, derived from incomplete oxidation of linoleic acid. In spite of dietary therapy, the patient died of respiratory acidosis at four months of age. Samples of liver and muscle from the autopsy were assayed for 2,4-dienoyl-coenzyme A reductase activity. Using the substrate 2-trans,4-cis-decadienoylcoenzyme A, the reductase activity was 40% of the control value in liver and only 17% of that found in normal muscle. It is suggested that unsaturated substrates should be used for in vitro testing to cover the full range of potential beta-oxidation defects and that acylcarnitine species identification be used for in vivo detection of this disorder.

A prospective randomized trial of itraconazole vs fluconazole for the prevention of fungal infections in patients with acute leukemia and hematopoietic stem cell transplant recipients.

Fluconazole antifungal prophylaxis is standard care in allogeneic hematopoietic stem cell transplant (HSCT) recipients, but this drug lacks anti-Aspergillus activity, the primary cause of invasive fungal infection (IFI) in many transplantation centers. We performed a randomized trial to compare itraconazole vs fluconazole, for prevention of IFIs in patients with acute leukemia (AL) and HSCT recipients. One hundred and ninety-five patients were randomly assigned to either fluconazole or itraconazole antifungal prophylaxis, after stratification into high-risk and low-risk groups. Antifungal prophylaxis was started at the beginning of chemotherapy and continued until resolution of neutropenia, or until amphotericin B treatment was started. IFI occurred in 11 (11%) of itraconazole, and in 12 (12%) fluconazole recipients. Invasive candidiasis (IC) developed in two (2%) itraconazole and one (1%) fluconazole recipients, while invasive aspergillosis (IA) developed in nine (9%) itraconazole and 11(11%) fluconazole recipients. There was no difference in the incidence of total IFI, IC and IA between the two study arms. However, there was a nonsignificant trend towards reduced mortality among patients who developed IA while receiving itraconazole prophylaxis (3/9=33% vs 8/11=73%, P=0.095).

The metabolism of (n-3) and (n-6) fatty acids and their oxygenation by platelet cyclooxygenase and lipoxygenase.

Arachidonic acid is the principal unsaturated acid in most membrane lipids. Membrane lipids also contain a variety of other (n-6) and (n-3) fatty acids. The amounts of (n-6) and (n-3) fatty acids in membrane lipids can be modified by dietary fat change. Our studies show that long chain (n-6) and (n-3) acids are metabolized by platelet lipoxygenase and cyclooxygenase. When cells are exposed to various agonists, a variety of unsaturated fatty acids may be released. Our studies show that they have the potential of modifying physiological function both by mediating arachidonic acid metabolism and as direct precursors for oxygenated metabolites which themselves may interact with specific receptors to regulate biological processes.

A comparison of the specific activities of linoleate and arachidonate in liver, heart and kidney phospholipids after feeding rats ethyl linoleate-9,10,12,13-d4.

In order to determine how dietary linoleate is metabolized, rats were maintained on a chemically defined diet containing 1.6% ethyl linoleate. After 5 weeks the linoleate was replaced by an equal amount of ethyl 9,10,12,13-d4-linoleate. The animals were killed 3 days later and the molar percentage of d4-linoleate and d4-arachidonate were quantified in liver, heart and kidney phospholipids. In liver, 54 and 22.8 mol% respectively of the esterified linoleate and arachidonate was deuteriated. The lower specific activity of arachidonate versus linoleate suggests that desaturation of linoleate, by a 6-desaturase, is not only rate limiting for synthesis of arachidonate but that the amount of newly synthesized arachidonate is insufficient by itself to maintain steady state levels of esterified arachidonate. The molar fraction of deuteriated linoleate in heart and kidney phospholipids was respectively 35 and 37.4%. These values are lower than for liver phospholipids but it appears there is adequate dietary linoleate available in these tissues for the synthesis of arachidonate. However, of the esterified arachidonate in heart and kidney phospholipids only 4.2 and 8.6 mol% respectively was deuteriated. Our results suggest that arachidonate is made in liver and transported to heart and kidney.

On the mechanistic reasons for the dual positional specificity of the reticulocyte lipoxygenase.

A set of octadecadienoic acid isomers and selected eicosatrienoic acids were tested as substrates for the lipoxygenases from soybeans and reticulocytes. Among the dienoic fatty acids, 8Z,11Z-octadecadienoic acid containing a n - 9 doubly allylic methylene group turned out to be the best substrate for the reticulocyte enzyme. This substrate was converted to its corresponding n - 7 hydroperoxy derivative. The soybean lipoxygenase, in contrast, prefers the 9Z,12Z-octadecadienoic acid (linoleic acid) which is oxygenated to its n - 6 hydroperoxy derivative. In both cases a strong preference for the LS-isomer has been observed. Analysis of the oxygenation products formed from various eicosatrienoic acids indicated that 8Z,11Z,14Z-eicosatrienoic acid was converted by the reticulocyte enzyme to its 12S- and 15S-hydroperoxy derivative in a ratio of about 1:7 (dual positional specificity), whereas the 7Z,10Z,13Z-isomer was oxygenated predominantly (greater than 97%) to its 14S-hydroperoxy derivative (singular positional specificity). 9Z,12Z,15Z-eicosatrienoic acid was oxygenated with a dual positional specificity to the corresponding 13- and 16-hydroperoxy compounds in a ratio of about 7:1. The soybean lipoxygenase converts the 8Z,11Z,14Z-isomer with a singular positional specificity to the corresponding 15S-hydroperoxy derivatives. The 9Z,12Z,15Z-eicosatrienoic acid, however, was oxygenated with a dual positional specificity to its 13S-hydroperoxy and 16S-hydroperoxy derivative in a ratio of about 1:4.

Metabolism of 6,9,12-octadecatrienoic acid and 6,9,12,15-octadecatetraenoic acid by rat hepatocytes.

When 5.10(6) hepatocytes were incubated for 40 min with 0.015-0.3 mM (1-14C)-labeled 6,9,12-octadecatrienoic acid or (1-14C)-labeled 6,9,12,15-octadecatetraenoic acid there was a concentration-dependent acylation of radioactive metabolites into both phospholipids and triacylglycerol. However, when the concentration of either substrate exceeded 60-150 microM there was no further increase in the metabolism of either substrate to longer-chain (n-6) and (n-3) acids. When cells were then incubated for various periods of time with 60 microM substrate there was initial rapid removal of the substrate which was accompanied by its acylation into lipids. Over time, the amount of both substrates in lipids declined without an overall drop in specific activity. This decline was accompanied by an increase in long-chain (n-6) and (n-3) fatty acids. Similar results were obtained when the time-dependent metabolism of the two substrates was examined in individual hepatocyte phospholipids. Collectively, these findings suggest that when these two 18-carbon acids are produced by desaturation of dietary linoleate and linolenate that they are in part initially acylated into a labile phospholipid pool. Rapid release and subsequent further metabolism to longer-chain (n-6) and (n-3) acids may explain why these products of the 6-desaturase do not accumulate in membrane lipids.

Metabolism of long-chain polyunsaturated fatty acids in isolated cardiac myocytes.

The uptake and integrated intracellular metabolism of (n - 6) and (n - 3) polyunsaturated fatty acids was studied in isolated rat cardiac myocytes and in the perfused heart. Labeled linolenic acid (18:3(n - 3)) uptake and its subsequent metabolism into carbon dioxide as well as acylation into lipids was nonsaturable over a substrate range of 0.02 to 0.4 mM. [1-14C]Linoleic acid (18:2(n - 6)), dihomo-gamma-linolenic acid (20:3(n - 6)) and arachidonic acid (20:4(n - 6)) were transported into myocytes at rates similar to those for linolenic acid. Conversely both [1-14C]-gamma-linolenic acid (18:3(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were taken up at a slower rate. Oxidation of 18:3(n - 6) was 4-5-fold greater when compared with C18-C20 polyunsaturated fatty acids. When myocytes were incubated with labeled 18:2(n - 6), 18:3(n - 6), 18:3(n - 3), 20:4(n - 6) or 20:5(n - 3), it was not possible to detect any desaturation or chain-elongation products. Identical results were obtained when hearts were perfused with 1-14C-labeled linoleic acid.

Metabolism of polyunsaturated fatty acids by an (n - 6)-lipoxygenase associated with human ejaculates.

Washed cells of normal human ejaculates were incubated with [14C]arachidonic acid (20:4(n - 6] at 37 degrees C for 30-40 min and the main product was characterized as 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid by reverse phase, straight phase and chiral phase high performance liquid chromatography (HPLC) and by capillary gas chromatography-mass spectrometry. The biosynthesis of 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid from exogenous 20:4(n - 6) was inhibited by nordihydroguaiaretic acid and abolished by heat inactivation, but it appeared to be unaffected by the ionophore A23187 and Ca2+. Human spermatozoa were partly purified from contaminating material by the swim-up procedure and incubated with 14C-labelled 18:2(n - 6), 20:4(n - 6), 22:5(n - 6) and 22:6(n - 3) for 30-40 min at 37 degrees C. The main radiolabelled products, which were obtained in low yields, co-chromatographed with the Ls (n - 6)-hydroxy fatty acid of each substrate on reverse phase, straight phase and chiral phase HPLC. The (n - 6)-lipoxygenase was also present in ejaculates with oligozoospermia or azoospermia. The seminal fluid contains membrane-surrounded organelles (e.g., 'prostasomes' secreted by the prostate gland) and the (n - 6)-lipoxygenase was present and appeared to be relatively prominent in almost cell-free preparations of organelles of seminal fluid. The (n - 6)-lipoxygenase activity associated with the spermatozoa may thus be explained by the presence of prostasomes or other organelles, which may conceivably bind to the spermatozoon through hydrophobic interactions.

Differences in the interconversion between 20- and 22-carbon (n - 3) and (n - 6) polyunsaturated fatty acids in rat liver.

When male weanling rats were fed diets containing either 5% corn oil or a diet in which half of the corn oil was replaced by fish oil, the 20:5(n - 3) in liver choline and ethanolamine phosphoglycerides, not only partially replaced arachidonate but also paired with palmitic and stearic acids in the same molar ratio as did arachidonate. The 22:5(n - 3)/22:6(n - 3) ratio in the liver phospholipids of corn oil fed rats was similar to that found when the esterified levels of these two acids were increased 5-fold by feeding fish oil. Moreover, the pairing of both 22:5(n - 3) and 22:6(n - 3) with palmitic and stearic acids, on a molar ratio basis, was relatively independent of the total amount of esterified 22:5(n - 3) and 22:6(n - 3). When (3-14C)-labeled 22:4(n - 6) was injected into rats raised on a chow diet or incubated with hepatocytes from these animals, its primary metabolic fate was retroconversion to arachidonate followed by esterification. Conversely, [3-14C]22:5(n - 3) was a poorer substrate for retroconversion with a larger amount being esterified directly into phospholipids and, in addition, this acid served as a precursor for 22:6(n - 3). The enhanced metabolism of both [3-14C]22:4(n - 6) to 22:5(n - 6) and of [3-14C]22:5(n - 3) to 22:6(n - 3) in animals raised on a diet devoid of fat or in their hepatocytes may possibly be due to elevated 6-desaturase activity and/or the level of this enzyme or enzymes. This hypothesis is based on studies showing that the synthesis of 22:6(n - 3) proceeds via a pathway independent of a 4-desaturase but requires the use of a 6-desaturase at two steps (Voss, A., Reinhart, M., Sankarappa, S. and Sprecher, H. (1991) J. Biol. Chem. 266, 19995-20000).

Metabolism of 8,11,14,17-eicosatetraenoic acid by human platelet lipoxygenase and cyclooxygenase.

Human platelets metabolize 8,11,14,17-eicosatetraenoic acid primarily into 12-hydroxy-8,10,14,17-eicosatetraenoic acid. Several other hydroxy acids were also produced in small amounts via an indomethacin insensitive pathway. Platelet cyclooxygenase metabolized this acid only into 12-hydroxy-8,10,14-heptadecatrienoic acid. It was not possible to detect any cyclic products even though vesicular gland cyclooxygenase metabolizes this (n-3) acid to 17,18-dehydroprostaglandin E1 (Oliw, E.H., Sprecher, H. and Hamberg, M. (1986) J. Biol. Chem. 261, 2675-2683).

Studies on the incorporation and transacylation of various fatty acids in choline and ethanolamine-containing phosphoacylglycerol subclasses in human neutrophils.

The incorporation of eight 14C-labeled fatty acids into human neutrophil phospholipids was investigated and the results were expressed as percent of the total phospholipid associated 14C-labeled substrate incorporated after an initial 15 min labeling and a subsequent 2 h reincubation in fatty acid-free buffer. In all cases, the PC fraction accounted for more than 40% of the total phospholipid radioactivity. The inositol-containing phosphoacylglycerols were also labeled well by all the fatty acids except 22:6(n - 3) and 16:0; however, a greater percentage of [14C]22:6(n - 3) was found in PE than that of any other labeled fatty acid substrate. In all cases, most of the radioactivity in PC after 15 min was in the diacyl subclass. After 2 h, there was a shift of [14C]20:4(n - 6), [14C]20:5(n - 3), [14C]22:6(n - 3) and [14C]18:4(n - 4) into the ether-linked subclass. No such shift was observed for [14C]16:0 or [14C]18:2(n - 6) and, although there was an increase in the percent radioactive 20:3(n - 6) and 20:3(n - 9) in ether-linked PC after 2 h, the total radioactivity in this fraction remained low by comparison. A similar shift in label occurred in the plasmalogenic-linked PE subspecies in cells labeled with [14C]20:4(n - 6), [14C]20:5(n - 3) and [14C]22:6(n - 3).

Studies to determine if rat liver contains multiple chain elongating enzymes.

According to the revised pathways of polyunsaturated fatty acid biosynthesis three, rather than two acids, must be chain elongated for converting linoleate and linolenate, respectively, to 22:5(n-6) and 22:6(n-3) (Sprecher et al. (1995) J. Lipid Res. 36, 2471-2477). The present study was undertaken to determine whether microsomes contained chain-length specific chain-elongating enzymes and, secondly, whether reaction rates for any of these reactions might be rate limiting in the synthesis of 24:5(n-6) and 24:6(n-3), which are the immediate precursors of 22:5(n-6) and 22:6(n-3). Rates of total chain elongation products produced from both 18:4(n-3) and 20:5(n-3) were about 3 nmol/min/mg of microsomal protein while only about 0.5 nmol/min/mg of 24:5(n-3) plus 24:6(n-3) was synthesized from 22:5(n-3). The rate of 24:5(n-3) synthesis was similar to that for the desaturation of 24:5(n-3), at position 6, to yield 24:6(n-3) (Geiger et al. (1993) Biochim. Biophys. Acta 1170, 137-142). The results suggest that the last chain elongation step in unsaturated fatty acid biosynthesis may be equally regulatory in governing the synthesis of fatty acids as is desaturation at position 6. When an enzyme saturating level of [1-(14)C]18:4(n-3) was incubated with increasing amounts of 18:3(n-6) there was a decrease in the production [1-(14)C]20:4(n-3). In a similar way it was observed that 18:4(n-3) inhibited the chain elongation of [1-(14)C]18:3(n-6). Identical cross-over inhibitory studies, using 20:4(n-6) and 20:5(n-3), as well as 22:4(n-6) and 22:5(n-3) also suggested that microsomes contain chain length specific chain-elongating enzymes. This conclusion was further supported by the finding that neither 20:5(n-3) or 22:5(n-3) inhibited the chain elongation of [1-(14)C]18:4(n-3). However, 18:4(n-3), and to a lesser degree, 22:5(n-3) did inhibit the chain elongation of [1-(14)C]20:5(n-3). This latter finding suggests that 18:4(n-3) and 20:5(n-3) might interact with the enzyme that chain elongates 20:5(n-3) to depress its ability to synthesize 22:5(n-3). Our results are most consistent with the presence of multiple chain-elongating enzymes, but a more definitive answer requires the purification of these membrane-bound proteins. In addition our results suggest that the channeling of acids between enzymes in the endoplasmic reticulum may play an important role in regulating the biosynthesis of unsaturated fatty acids.

The isolation of acyl-CoA derivatives as products of partial reactions in the microsomal chain elongation of fatty acids.

An analysis of overall chain elongation, condensation, beta-hydroxyacyl-CoA dehydrase and 2-trans enoyl-CoA reductase reactions, using the appropriate CoA derivatives as substrates which are required in the microsomal chain elongation of both palmitoyl-CoA and 6,9-octadecadienoyl-CoA, demonstrated that in each instance, the products of these reactions were the CoA derivatives. Reverse dehydrase reactions run with 2-trans enoyl-CoA derivatives as substrates, in the absence of NADPH, revealed that the product was the beta-hydroxyacyl-Coa. In the presence of NADPH, incubations with beta-hydroxyacyl-CoA demonstrated that both the 2-trans derivatives and the alpha, beta-saturated product were recovered as their CoA derivatives. These latter findings are more consistent with the involvement of discrete dehydrase and 2-trans-enoyl-CoA reductase enzymes rather than a single protein catalyzing two reactions.