Potential inhibitors of rat tooth alkaline phosphatase studied by means of different histochemical techniques.

Two histochemical methods for demonstration of alkaline phosphatase activity, a lead pyrophosphate- anda naphtholphosphate technique, were compared. Since different results may be due to methodological differences as well as different enzyme activities, the enzymatic hydrolysis of the naphtholphosphate was visualized both by means of an azo-dye coupler and by lead-capturing of the liberated phosphate ion. Various potential inhibitors of alkaline phosphatase activity (diphosphonate, D-penicillamine, and sodium fluoride) were also tested. The use of diphosphonate and D-penicillamine resulted in inhibited or reduced staining, which could mainly be explained by an interference by these compounds with components in the incubation media rather than with the enzyme itself. The addition of sodium fluoride had no effect on the naphtholphosphate staining pattern irrespective of capturing method, whereas the odontoblastic pyrophosphate splitting alkaline phosphatase appeared to be sensitive to sodium fluoride, suggesting the presence of two alkaline phosphatases in odontoblasts.     

D-Penicillamine: analysis of the mechanism of copper-catalyzed hydrogen peroxide generation

Recent studies have suggested that the inhibition of lymphocyte mitogenesis by D-penicillamine in the presence of copper could be mediated by the formation and action of hydrogen peroxide. To explore this possibility further, we first sought evidence of H2O2 generation by D-penicillamine in a cell-free system by a) measurement of copper-catalyzed D-penicillamine oxidation and the requirement for oxygen in this process; b) direct measurement of H2O2 formation during D-penicillamine oxidation by the peroxidase-mediated oxidation of fluorescent scopoletin; and c) evaluation of the possible synthesis of O2- during D-penicillamine oxidation. The addition of copper to D-penicillamine in physiologic buffer catalyzed D-penicillamine oxidation in a dose-dependent fashion. D-penicillamine oxidation was accompanied by O2 consumption with a molar ratio of approximately 2:1, but did not occur under anaerobic conditions. Furthermore, D-penicillamine oxidation resulted in the formation of amounts of H2O2 stoichiometrically equivalent to oxygen consumption (i.e., 1:1). Copper-catalyzed D-penicillamine oxidation caused reduction of nitroblue tetrazolium in a reaction blocked by superoxide dismutase, suggesting the formation of O2-. Additional studies confirmed that D-penicillamine inhibited PHA-induced mitogenesis of lymphocytes in the presence of copper, and that catalase protected the cells from this action. Furthermore, when polymorphonuclear leukocytes were incubated with D-penicillamine plus copper, hexose monophosphate shunt activity increased up to threefold with abrogation of this stimulation by catalase. None of the effects of D-penicillamine plus copper on cells were diminished by hydroxyl radical scavengers mannitol or benzoate. These results are consistent with oxygen-dependent copper-catalyzed oxidation of D-penicillamine in aqueous solutions leading to the formation of O2- and H2O2. H2O2 produced by this reaction can inhibit lymphocyte mitogenesis and stimulate neutrophil hexose monophosphate shunt activity in vitro and may be relevant to the therapeutic effects of D-penicillamine in vivo.     

D-penicillamine-induced granulomatous hepatitis in brown Norway rats

The mechanism of idiosyncratic drug reactions (IDRs) remains poorly understood. D-penicillamine treatment is associated with a wide range of autoimmune reactions including liver injury. An animal model which utilizes brown Norway (BN) rats has been used to investigate the mechanism of D-penicillamine-induced IDRs because it mimics the autoimmune reactions that occur in humans. The purpose of this study was to investigate the type of liver injury that results from D-penicillamine treatment in BN rats. We had previously noted that D-penicillamine caused histological changes in the liver, but there was no increase in alanine transaminase (ALT), and we assumed that there was no significant injury. However, we subsequently discovered that D-penicillamine inhibits the ALT assay. In the present study, we found that treatment of BN rats with a low doses of D-penicillamine (10 or 15 mg/day) resulted in a mild increases in glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities; however, this was not associated with histological changes. A higher dose of D-penicillamine (20 mg/day) resulted in 63 % of the rats developing a skin rash, and these rats had elevated serum GLDH and SDH levels with histopathological changes characteristic of granulomatous hepatitis. This included large clusters of leukocytes in the form of granulomas that contained neutrophils, macrophages, and CD8 T cells. These changes did not occur in the rats that did not get sick. This model may be a good model to investigate the characteristics of drug-induced granulomatous hepatitis.     

The effect of D-penicillamine on myeloperoxidase: formation of compound III and inhibition of the chlorinating activity

The inhibitory effect of the anti-arthritic drug D-penicillamine on the formation of hypochlorite (HOCl) by myeloperoxidase from H2O2 and Cl- was investigated. When D-penicillamine was added to myeloperoxidase under turnover conditions, Compound III was formed, the superoxide derivative of the enzyme. Compound III was not formed when D-penicillamine was added in the presence of EDTA or in the absence of oxygen. However, when H2O2 was added to myeloperoxidase, D-penicillamine and EDTA, Compound III was formed. Therefore it is concluded that formation of Compound III is initiated by metal-catalysed oxidation of the thiol group of this anti-arthritic drug, resulting in formation of superoxide anions. Once Compound III is formed, a chain reaction is started via which the thiol groups of other D-penicillamine molecules are oxidized to disulphides. Concomitantly, Compound I of myeloperoxidase would be reduced to Compound II and superoxide anions would be generated from oxygen. This conclusion is supported by experiments which showed that formation of Compound III of myeloperoxidase by D-penicillamine depended on the chloride concentration. Thus, an enzyme intermediate which is active in chlorination (i.e. Compound I) participated in the generation of superoxide anions from the anti-arthritic drug. From the results described in this paper it is proposed that D-penicillamine may exert its therapeutic effect in the treatment of rheumatoid arthritis by scavenging HOCl and by converting myeloperoxidase to Compound III, which is inactive in the formation of HOCl.     

The effect of D-penicillamine on human myeloperoxidase, a mechanism for the efficacy of the drug in rheumatoid arthritis

We investigated the effect of D-penicillamine on the ability of myeloperoxidase, purified from human leukocytes, to catalyse the oxidation of chloride ions to hypochlorite (HOCl) in the presence of H2O2. It is shown that, due to the interaction of D-penicillamine with both myeloperoxidase itself and HOCl, the chlorinating activity of myeloperoxidase in the presence of H2O2 and chloride ions is prevented. A concentration of 100 microM D-penicillamine inhibits the chlorinating activity of myeloperoxidase completely, which Is due to the stabilization of Compound II, an inactive form of the enzyme. In addition, HOCl reacts directly with D-penicillamine. Analysis of the reaction products of D-penicillamine and HOCl showed that D-penicillamine was oxidized to penicillamine disulphide and penicillamine sulphinic acid, and eventually deaminated (indicated by the release of ammonia). Lower concentrations of D-penicillamine (10 microM) inhibited myeloperoxidase less, but still acted as effective scavengers of HOCl. In very low concentrations (1 microM), D-penicillamine did not scavenge HOCl effectively, but rather stimulated the chlorinating activity of myeloperoxidase. However, when instead of D-penicillamine a comparable amount of ascorbate was added, a similar but even larger stimulation was observed. Since the concentration of free D-penicillamine in serum from rheumatoid patients treated with this drug is about 20 microM (Saetre, R. and Rabenstein, D.L. (1978) Anal. Chem. 50, 276-280), the therapeutic effect of D-penicillamine may be due to the protection of tissues against the reactive HOCl released by activated granulocytes at inflammation sites.     

Effects of D-penicillamine on the ultrastructure of hepatocytes and state of the base substance in the liver of rats with experimental cirrhosis

The authors examined the action of D-penicillamine on the ultrastructure of hepatocytes and the condition of the base substance in the rat's liver, with experimental CCl4-cirrhosis. (D-penicillamine was given to these rats during 4 and 6 months). It was discovered that the using of D-penicillamine on the early stages of experiment (until 4 months) reduced the process of the development of the liver cirrhosis. This fact was confirmed by the reducing of the level of base substance in the liver and reducing of the quantity of collagen in the Disse space, as well as by the absence of fibres in the intercellular spaces near sinusoid. When the D-penicillamine was given longer the increasing of the beta-NAG activity with simultaneous severe reducing of the GAG and destruction of hepatocyte's organelles in the experimental rat to the 6 months of experiment were observed. The authors consider that these data are the evidence of the negative effect of the long using of D-penicillamine in the stage of decompensated cirrhosis of the liver.     

Collagen cross-linking. Effect of D-penicillamine on cross-linking in vitro.

D-Pencillamine is believed to inhibit collagen cross-link biosynthesis by forming thiazolidine rings with lysyl-derived aldehydes that are intermediates in bifunctional cross-link synthesis. Recently, we showed that aldehyde biosynthesis catalyzed by lysyl oxidase occurs after the onset of fibril formation and that nascent aldehydes form Schiff-base cross-links rapidly in fibrils. This suggested that the accessibility of D-penicillamine to most aldehydes formed during cross-link synthesis might be limited. To study this, reconstituted chick bone collagen fibrils were incubated in vitro with highly purified lysyl oxidase and D-penicillamine. As reported in previous studies in vivo, allysine content increased and polyfunctional cross-link synthesis decreased with D-penicillamine. However, the concentration of bifunctional cross-links increased rather than decreased due to a 2-fold increase in N6:6'-dehydro-5,5'-dihydroxylysinonorleucine. Hydroxyallysine, an intermediate in formation of this Schiff base, decreased. A time study indicated that allysine levels increased primarily after the bulk of Schiff base synthesis. These results indicate that D-penicillamine does not inhibit bifunctional cross-link synthesis as previously suggested. Its principal effect is to block synthesis of polyfunctional cross-link products from Schiff base cross-link precursors and to cause accumulation of these precursors. This effect may be due to interference with the close molecular packing required for polyfunctional cross-link synthesis. These results also suggest a mechanism for the relative insensitivity of tissues such as bone with high hydroxylysine content to D-penicillamine. In this study, D-penicillamine caused selective accumulation of allysyl and not hydroxyallysyl residues. In bone as opposed to soft tissues, hydroxyallysyl residues are intermediates in synthesis of almost all cross-links.     

Ribosome-inactivating proteins from plants: more than RNA N-glycosidases?

Many plants contain proteins that are capable of inactivating ribosomes and accordingly are called ribosome-inactivating proteins or RIPs. These typical plant proteins receive a lot of attention in biological and biomedical research because of their unique biological activities toward animal and human cells. In addition, evidence is accumulating that some RIPs play a role in plant defense and hence can be exploited in plant protection. To understand the mode of action of RIPs and to optimize their medical and therapeutical applications and their use as antiviral compounds in plant protection, intensive efforts have been made to unravel the enzymatic activities of RIPs and provide a structural basis for these activities. Though marked progress has been made during the last decade, the enzymatic activity of RIPs has become a controversial issue because of the concept that RIPs possess, in addition to their classical RNA N-glycosidase and polynucleotide:adenosine glycosidase activity, other unrelated enzymatic activities. Moreover, the presumed novel enzymatic activities, especially those related to diverse nuclease activities, are believed to play an important role in various biological activities of RIPs. However, both the novel enzymatic activities and their presumed involvement in the biological activities of RIPs have been questioned because there is evidence that the activities observed are due to contaminating enzymes. We offer a critical review of the pros and cons of the putative novel enzymatic activities of RIPs. Based on the available data, it is suggested that there is little conclusive evidence in support of the presumed activities and that in the past too little attention has been given to the purity of the RIP preparation. The antiviral activity and mode of action of RIPs in plants are discussed in view of their classical and presumed novel enzymatic activities.     

An investigation into copper catalyzed D-penicillamine oxidation and subsequent hydrogen peroxide generation

D-Penicillamine is a potent copper (Cu) chelating agent. D-Pen reduces Cu(II) to Cu(I) in the process of chelation while at the same time being oxidized to D-penicillamine disulfide. It has been proposed that hydrogen peroxide is generated during this process. However, definitive experimental proof that hydrogen peroxide is generated remains lacking. Thus, the major aims of these studies were to confirm and quantitatively assess the in vitro production of hydrogen peroxide during copper catalyzed D-penicillamine oxidation. The potential cytotoxic effect of hydrogen peroxide generation was also investigated in vitro against MCF-7 human breast cancer cells. Cell cytotoxicity resulting from the incubation of D-penicillamine with copper was compared to that of D-penicillamine, copper and hydrogen peroxide. The mechanism of copper catalyzed D-penicillamine oxidation and simultaneous hydrogen peroxide production was investigated as a function of time, concentration of cupric sulfate or ferric chloride, temperature, pH, anaerobic condition and chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid. A simple, sensitive and rapid HPLC assay was developed to simultaneously detect D-penicillamine, its major oxidation product D-penicillamine disulfide, and hydrogen peroxide in a single run. Hydrogen peroxide was shown to be generated in a concentration dependent manner as a result of D-penicillamine oxidation in the presence of cupric sulfate. Chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid were able to inhibit D-penicillamine oxidation. The incubation of MCF-7 human breast cancer cells with D-penicillamine plus cupric sulfate resulted in the production of reactive oxygen species within the cell and cytotoxicity that was comparable to free hydrogen peroxide.     

Adverse reactions to D-penicillamine after gold toxicity.

The incidence of adverse reactions to D-penicillamine in 155 patients with rheumatoid arthritis was analysed and compared with their history of adverse reactions to gold. Out of 125 patients who took only D-penicillamine, 45 developed side effects from the drug, whereas of 27 patients with a history of gold toxicity, 18 also reacted adversely to D-penicillamine. All patients who took D-penicillamine within six months after an adverse reaction to gold developed side effects from D-penicillamine. Fourteen patients developed similar adverse reactions to D-penicillamine and gold, and the interval between treatments in this group was significantly shorter (p less than 0.01) than in those who developed either differing adverse reactions to both drugs or no reaction to D-penicillamine after treatment with gold. An interval exceeding six months between treatment with gold and treatment with D-penicillamine in patients who have developed adverse reactions to gold apparently reduces the risk of adverse reactions to D-penicillamine.     

Platelet aggregation and thromboxane A2 release in primary biliary cirrhosis and effect of D-penicillamine treatment

Platelet function was assessed in 28 patients with primary biliary cirrhosis (PBC), of whom 10 were receiving D-penicillamine. Patients not on D-penicillamine treatment had platelet aggregation similar to that in the healthy control group; the group treated with D-penicillamine showed significantly enhanced platelet aggregation in response to threshold doses of adrenaline and collagen but not ADP. Median thromboxane B2 production was also higher in D-penicillamine treated patients than in controls or untreated patients; this difference did not reach statistical significance. The addition of D-penicillamine in vitro to platelet rich plasma from normal subjects was shown to enhance adrenaline- and collagen-induced platelet aggregation. Abnormalities of platelet function in PBC patients did not correlate with serum cholesterol concentration or with liver function tests but were related to the stage of disease. The present study emphasises the need to consider the aetiology, disease stage and type of treatment when assessing platelet function and prostanoid release in liver disease.     

Effect of monothiol along with antioxidant against mercury-induced oxidative stress in rat

Efficacy of thiol chelators viz. N-acetyl cysteine and D-penicillamine (NAC and DPA) along with nutritional supplements viz. zinc acetate, sodium selenite and magnesium sulphate (Zn, Se and Mg) in the treatment of mercury intoxication was investigated in rats. This is of particular interest since high bonding affinity between mercuric ion and the thiol group exits. The mutual antagonism of mercury and selenium is one of the strongest examples of the interaction in the trace element field. Adult rats of Sprague-Dawley strain were administered a bolus dose of dimethyl mercury (10 mg/kg) orally. A significant rise in the aspartate aminotransferase, alanine aminotransferase, serum alkaline phosphatase, lactate dehydrogenase, gamma glutamyltranspeptidase, bilirubin and creatinine were observed. Single mercury exposure also resulted in a significant increase in lipid peroxides with a concomitant decrease in reduced glutathione level in liver, kidney and brain. A decrease in the enzymatic activities of acetyl cholinesterase in different regions of the brain was observed. These parameters were restored considerably with chelating agents along with nutritional supplementation, but NAC+Se and DPA+Mg offered significant protection in comparison with other combinations.     

Extracellular activity in Cryptococcus neoformans strains isolated from AIDS patients and from environmental sources

Nineteen Cryptococcus neoformans strains isolated from AIDS patients and 16 from bird droppings were tested for their extracellular activity. Typical enzymatic activity that was different from other medically important yeasts was found. The results obtained may indicate that there are new extracellular enzymatic activities that imply a relationship between C. neoformans and its virulence. A correlation among the different enzymatic activities was also investigated and according to the results obtained no relationship was observed among any of the recorded extracellular enzymatic activities. Research on C. neoformans extracellular enzymatic activity is useful not only to better understand its metabolism but in particular to establish a possible relationship between its virulence and pathogenicity.     

D-penicillamine inhibits transactivation of human immunodeficiency virus type-1 (HIV-1) LTR by transactivator protein

D-Penicillamine, an amino acid analogue of cysteine, has been shown to inhibit the transactivation of HIV-1 LTR by the transactivator protein, tat protein. The transactivation was studied in Jurkat cells co-transfected with plasmids containing HIV-LTR sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and HIV tat gene. The expression of CAT activity was a measure of transactivation of LTR by the tat protein. Incubation of transfected Jurkat cells with D-penicillamine led to inhibition of CAT activity. This inhibition was found to be concentration-dependent; more than 90% inhibition of chloramphenicol acetylation was seen in extracts prepared from cultures incubated with 40 micrograms/ml of D-penicillamine. Earlier experiments have shown that D-penicillamine at 40 micrograms/ml can completely inhibit HIV-1 (HTLV-III B) replication in H9 cells [(1986) Drug Res. 36, 184-186]. These results suggest that inhibition of transactivation may be the molecular mechanism involved in the inhibition of HIV-1 replication by D-penicillamine.     

Geographical and climatic differences in long-term effect of organic and inorganic amendments on soil enzymatic activities and respiration in field experimental stations of China

Soil enzymatic activities and basal respiration are two important bio-indicators of soil quality and are closely related to soil fertility and sustainable crop growth. We collected soil samples from four long-term experimental stations of China differing both geographically and climatically – Shenyang Agro-Ecological Experimental Station (brown soil), Fengqiu State Key Agro-Ecological Experimental Station (fluvo-aquic soil), Taoyuan Agro-Ecological Experimental Station (paddy soil) and Qiyang Red Soil Experimental Station (red soil) – to investigate the effect of long-term application of chemical fertilizer and/or organic manure on soil enzymatic activities and basal respiration. In general, soil enzymatic activities and basal respiration increased in soils with long-term application of chemical fertilizers and manures, especially organic manure. Among stations, soil enzymatic activities were not consistently increased with organic manure alone (e.g. the sucrase and phosphatase activities in the paddy soil from Taoyuan and red soil from Qiyang). As well, soil enzymatic activities were characterized by distinct zonality. Soil cellulase and urease activity were the two main indicators of soil fertility as evaluated by principal component analysis. Regression analysis of the major abiotic factor(s) influencing enzyme activity revealed a dependence on the specific enzyme tested and the activity of specific soil enzymes depended on the location of the experimental station. Environmental factors, mainly soil temperature, moisture and pH, as well as soil texture, should be taken into account in evaluating the effects of long-term fertilization on soil biological activity and soil fertility in field experimental stations differing geographically and climatically.     

NADH/NADPH--cytochrome c reductase assay: interference by nonenzymatic cytochrome c reducing activity in plant extracts

Considerable thermostable, very probably nonenzymatic, cytochrome c reducing activities are present in all plant extracts studied. These interfere with the assays of NADH- and NADPH-cytochrome c reductases. Since this could be a widespread, but scarcely considered phenomenon, a method was looked for to avoid this problem. Because of the low-molecular-weight nature of the compounds exhibiting the thermostable activity, separation of enzymatic and nonenzymatic activities is achieved by Sephadex G-25 filtration. This separation allows the optimization of the assay of antimycin A-insensitive NADH-cytochrome c reductase of maize root tips. The enzymatic and nonenzymatic activities are shown to differ in many respects (the nonenzymatic activity is not significantly NADH-dependent), but even in the optimized conditions, the nonenzymatic activity is not eliminated totally if homogenates are tested without previous Sephadex filtration. So the separation of the different active compounds on Sephadex G-25 seems to be a prerequisite for obtaining exact data on enzymatic cytochrome c reducing activities in plant extracts.     

Complexes of sulfur-containing ligands. I. Factors influencing complex formation between D-penicillamine and copper (II) ion.

Complex formation and redox reactions between copper (II) ion and D-penicillamine were studied in detail as functions of the metal/-ligand ratio and the concentration of halide ions. It was established that a copper (I)- D-penicillamine polymeric complex of amphoteric character is formed when excess D-penicillamine is present. When the D-penicillamine/copper (II) ratio = 1.45 in the starting reaction mixture, a mixed valence complex with an intense red-violet color is formed. The formation of this compound, which contains 44% copper (II) ion, is greatly influenced by the experimental conditions, primarily by the concentration of halide ions. The main chemical and physical characteristics of the mixed valence complex were determined via magnetic and spectroscopic measurements. It was further established that a very intense blue complex is formed when the D-penicillamine/copper (II) ratio = 2 and halide ions are present. On the basis of the nature of the products formed under various conditions it was concluded that the copper (II)-D-penicillamine system may serve as a good model for studying the binding sites of copper-containing proteins.     

6-Sulfanilamidoindazole arthritis: Influence of alloxan diabetes,phenylbutazone and D-penicillamine on inflammatory size,sulfhydryl groups,diphenylamine reaction and enzymes of the serum

The influence of alloxan diabetes, phenylbutazone (20 mg/kg), and D-penicillamine (30 mg/kg) on 6-sulfanilamidoindazole arthritis was investigated. Arthritis was not altered by the alloxan-diabetic stage neither by injecting alloxan during developing arthritis nor by injecting it before the beginning of 6-sulfanilamidoindazole administration. However, the arthritis was completely suppressed by phenylbutazone after both methods of administration. D-penicillamine was without significant effect. Decrease of serum sulfhydryl groups and increase of serum diphenylamine value occurring during arthritis were prevented by phenylbutazone whereas alloxan and D-penicillamine did not.     

A comparative study of the biological properties of some sea snake venoms.

1. The protease, phosphodiesterase, alkaline phosphomonoesterase, L-amino acid oxidase, acetylcholinesterase, phospholipase A, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, procoagulant, anticoagulant and hemorrhagic activities of ten samples of venoms from seven taxa of sea snakes were examined. 2. The results show that venoms of sea snakes of both subfamilies of Hydrophiinae and Laticaudinae are characterized by a very low level of enzymatic activities, except phospholipase A activity and, for some species, hyaluronidase activity. 3. Because of the low levels of enzymatic activities and the total lack of procoagulant and hemorrhagic activities, venom biological properties are not useful for the differentiation of species of sea snakes. Nevertheless, the unusually low levels of enzymatic activities of sea snake venoms may be used to distinguish sea snake venoms from other elapid or viperid venoms.     

Effects of seasonal and decadal warming on soil enzymatic activity in a P‐deficient Mediterranean shrubland

Soil enzymes are central in the response of terrestrial ecosystems to climate change, and their study can be crucial for the models’ implementation. We investigated for 1 year the effects of warming and seasonality on the potential activities of five soil extracellular enzymes and their relationships with soil moisture, phosphorus (P) concentration, and other soil parameters in a P‐limited Mediterranean semiarid shrubland. The site was continuously subjected to warming since 1999, and we compared data from this study to analogous data from 2004. Warming uniformly increased all enzymes activities, but only when a sufficient amount of soil water was available. Seasonality unevenly altered enzyme activities, thus affecting enzymatic stoichiometry. P deficiency affected enzymatic stoichiometry, favoring the activities of the phosphatases. The effect of warming was stronger in 2014 than 2004, excluding the hypothesis of acclimation of rhizospheric responses to higher temperatures and suggesting that further increases in extracellular enzymatic activities are to be expected if sufficient water is available. Climatic warming will likely generally stimulate soil enzymatic activities and accelerate nutrient mineralization and similar ecological processes such as the production and degradation of biomass and changes in community composition, but which will be limited by water availability, especially in Mediterranean soils in summer. Winters in such ecosystems will benefit from a general increase in activity and production, but biological activity could even decrease in summer, potentially leading to a negative overall balance of nutrient mineralization. This study suggests that a general increase in activity due to warming could lead to faster mineralization of soil organic matter and water consumption in colder climates, until one of these factors in turn becomes limiting. Such trade‐offs between water and temperature in relation with enzyme activity should be considered in biogeochemical models.