2 Ecology of Microbial Enzymes in Lake Ecosystems Ryszard Jan Chro ´ st and Waldemar Siuda University of Warsaw, Warsaw, Poland I. INTRODUCTION During the past decade, an increasing number of ecological studies have considered the complexity of freshwater ecosystems. One major outcome of these studies has been an accelerated interest in the role of heterotrophic microorganisms (particularly bacteria) in the functioning of aquatic environments and the processes by which organic matter is made available to them (1–4). These heterotrophic microorganisms are the key trophic level at which the metabolism of the whole ecosystem is affected, i.e., organic matter decomposition, nutrient cycling, and structure of aquatic food webs. The demonstration of the importance of heterotrophic bacteria as a particulate carbon source for higher trophic levels and a major respiratory sink has created a renewed interest in the production and utilization of organic substrates by these microorganisms. Most organic compounds produced in natural waters have a polymeric structure (5,6) and they are too large to be readily assimilated. The transport of organic molecules across microbial cell membranes is an active process mediated by specific enzymes called permeases. Only the low-molecular-weight organic molecules (monomers or oligomers) can therefore be taken up (7). In order to be available for microbial metabolism, polymeric compounds must be transformed into smaller molecules through enzymatic depolymeriza- tion. Besides the physicochemical conditions of aquatic environments, the composition and availability of organic matter are the major factors that influence the development and activity of heterotrophic bacteria (8,9). The heterotrophic bacteria are the only biological populations capable of significantly altering both dissolved (DOM) and particulate (POM) organic matter. Microbial enzymes associated with these processes are the principal cata- lysts for a large number of biochemical transformations of organic constituents in aquatic environments. Many of these transformations can be mediated only by heterotrophic bac- teria because the enzyme systems required for these reactions are not found in other or- ganisms. Microorganisms have adopted essentially two strategies that enable them to utilize macromolecular compounds. The macromolecule can be engulfed by the cytoplasmic Copyright © 2002 Marcel Dekker, Inc. membrane to form a vacuole within the cytoplasm. Enzymes are secreted into this vacuole and the polymeric compounds are hydrolyzed and subsequently taken up. Uptake of sub- strate solutions by this method is referred to as pinocytosis; uptake of particulate substrates is termed phagocytosis. However, many microbial cells are unable to carry out these pro- cesses, and therefore pinocytosis and phagocytosis are restricted to only those eukaryotic microorganisms that lack a cell wall, e.g., many protozoa. Those eukaryotic and prokary- otic microorganisms that possess a cell wall have developed an alternative strategy for the assimilation of polymeric substrates. Hydrolytic enzymes are secreted outside the cyto- plasmic membrane, where they hydrolyze macromolecules in close vicinity to the cell. The resulting low-molecular-weight products are then transported across the cell mem- brane and utilized inside the cytoplasm. The hydrolysis of polymers is an acknowledged rate-limiting step in the utilization of organic matter by microorganisms in aquatic and soil environments. Prior to incorpora- tion into microbial cells, polymeric materials undergo stepwise degradation by a variety of cell surface–associated enzymes and/or enzymes secreted by intact living cells or liber- ated into the environment through the lysis of microorganisms. The importance of micro- bial enzymatic activities to the mobilization, transformation, and turnover of organic and inorganic compounds in freshwater and marine environments has been shown in many studies (10–18). Results of these studies have shown that studying enzymatic processes provides powerful information that helps in understanding basic processes of decomposi- tion and microbial activity in both freshwater and marine ecosystems. II. ORIGIN AND ASSOCIATION OF ENZYMES WITH AQUATIC MICROORGANISMS Three common terms are used for the enzymes involved in the transformation and degrada- tion of polymeric substrates outside the cell membrane: ectoenzymes (19), extracellular enzymes (20), and exoenzymes (21). In this chapter, the term ectoenzyme is used to refer to any enzyme that is secreted and actively crosses the cytoplasmic membrane and remains associated with its producer. Ectoenzymes are cell-surface-bound or periplasmic enzymes that react outside the cytoplasmic membrane with polymeric substrates that do not pene- trate the cytoplasm. Extracellular enzymes occur in free form dissolved in the water and/ or are adsorbed to surfaces (e.g., detrital particles, organic colloids, humic complexes, minerals in suspension). Extracellular enzymes in water may be secreted actively by intact viable cells, they can be released into the environment after cell damage or viral lysis, and/or they may result from zooplankton grazing on algal cells and from protozoan grazing on bacteria. Ectoenzymes and extracellular enzymes (in contrast to intracellular enzymes) react outside the cell, and most of them are hydrolases. The ectoenzymes that cleave polymers by splitting the key linkages on the interior of the substrate molecule and form intermediate sized fragments are called endoectoenzymes (e.g., aminoendopeptidases act on the cen- trally located peptide bonds and liberate peptides) (22). Those ectoenzymes that hydrolyze the substrate by consecutive splitting of monomeric products from the end of the molecule are termed exoectoenzymes (e.g., aminoexopeptidases hydrolyze peptide bonds adjacent to terminal α-amino or α-carboxyl groups and liberate free amino acids) (23). There are three pools of microbial enzymes in water samples: intracellular enzymes are located and react with substrates inside the cytoplasmic region and are mostly responsi- Copyright © 2002 Marcel Dekker, Inc. Figure1Percentagedistributionofcell-boundandextracellularactivityofmicrobialchitinase (CHTase),deoxyribonuclease(DNase),5′-nucleotidase(5′-nase),alkalinephosphatase(APase),β- glucosidase(GLCase),andaminopeptidase(AMPase)inwatersamplesfromeutrophicLakeMi- kołajskie.(Chro ´ st,unpublished.) bleforinternalcellmetabolism;extracellularenzymesareinthesurroundingenvironment andcatalyzereactionswithoutcontrolfromtheirproducers;andectoenzymesarecell- surface-boundenzymes,mostlyhydrolases,thatdegradepolymericsubstrates,yielding readilyutilizablemonomers.Allpoolsarecomposedofbothendo-andexoenzymes. Distributionbetweenecto-andextracellularactivityforselectedenzymes(amino- peptidase,β-glucosidase,alkalinephosphatase[APase],5′-nucleotidase[5′-nase],deoxyri- bonuclease[DNase],andchitinase[CHTase])hasshownthatectoenzymescontributed onaveragefrom75%(APase)to98%(chitinase)ofthetotalactivityinlakewater(Fig. 1).Activitiesoftheintracellularandextracellularpoolenzymesarelow.Intracellular enzymescontributedfrom0.5%(chitinase)to10.7%(aminopeptidase)tothetotalactivity ofwatersamples.Activityoftheextracellularenzymes,dissolvedinthewater,constituted from1%(5′-nase)to16.5%(APase)ofthetotalactivity.Aninterestingobservationis thatextracellularenzymeactivityasapercentageoftotalactivityishigherinlakesedi- mentsthaninthewatercolumn(Table1).Thiswasparticularlyevidentinthecaseof chitinase and lipase activities. Enzyme activities bound to the 0.2- to 1.0-µm-size fraction of microplankton (mainly composed of bacteria) make up a greater fraction of activity by microorganisms in lake water. High ectoenzyme activity found in this size fraction has correlated with Copyright © 2002 Marcel Dekker, Inc. Table 1 Percentage Contribution of Extracellular Enzyme Activities to the Total Activity of Lake Water and Lake Sediment Samples Percentage Enzyme Lake/trophic status Water Sediment Leucine-aminopeptidase Plußsee/eutrophic 9.5 Ϯ 3.7 12.6 Ϯ 6.4 Scho ¨ hsee/mesotrophic 8.4 Ϯ 2.1 11.2 Ϯ 5.1 α-Glucosidase Plußsee/eutrophic 10.2 Ϯ 4.9 15.1 Ϯ 7.3 Mikołajskie/eutrophic 8.7 Ϯ 2.6 13.3 Ϯ 3.8 Szymon/hypereutrophic 10.8 Ϯ 3.3 14.6 Ϯ 4.9 Lipase Jagodne/hypereutrophic 28.8 Ϯ 9.6 41.0 Ϯ 7.4 Bełdany/eutrophic 21.4 Ϯ 6.8 33.1 Ϯ 8.3 Kisajno/mesotrophic 22.4 Ϯ 6.2 35.8 Ϯ 9.1 Alkaline phosphatase Plußsee/eutrophic 24.5 Ϯ 6.3 32.6 Ϯ 7.3 Chitinase Plußsee/eutrophic 2.3 Ϯ 0.7 22.8 Ϯ 6.9 Scho ¨ hsee/mesotrphic 1.7 Ϯ 0.5 23.2 Ϯ 8.4 Ϯ Standard deviation of an average value. Source: Data from Chro ´ st, unpublished. bacterial abundance and/or bacterial production of lake water. A variety of microorgan- isms produce ectoenzymes in waters and sediments in freshwater ecosystems. However, many studies have reported that bacteria are the major producers of ectoenzymes among aquatic microorganisms (24–33). III. CONTROL OF ECTOENZYME SYNTHESIS AND ACTIVITY The conditions in the aquatic environment, as in the soil aqueous phase, are unfavorable for enzymes. First, the substrate concentration is usually very low and highly variable. Many substrates may be insoluble, exist in intimate association with other compounds, and/or be bound to humic substances, colloidal organic matter, and detritus. Therefore, these conditions are suboptimal for the coupling of an enzyme to its substrate. Second, an enzyme may be lost from the parent cell and may be bound to suspended particles and humic materials, or it may be exposed to a variety of inhibitors present in the water. Finally, an enzyme may be denaturated by physical and chemical factors in the aquatic environment or hydrolyzed by proteases. Obviously, for an enzyme to be of benefit to its producer microorganism, it must avoid degradation long enough to associate with its substrate. Moreover, even if an enzyme overcomes these obstacles and binds with its substrate, the physical and chemical condi- tions of the reaction medium may be unsuitable for catalysis (e.g., nonoptimal pH or tem- perature, presence of inhibitors, absence of activators, suboptimal ionic strength). Never- theless, there is strong evidence that various aquatic microorganisms produce ectoenzymes in freshwaters that encounter a number of polymeric substrates (31) and that microbial growth is dependent on the products of ectoenzymatic reactions (13,14,16,24, 34–36). A microbial cell living in an aquatic ecosystem is influenced by a variety of environ- mental factors. The signal for appropriate gene expression and consequent ectoenzyme production within a cell is in response to the surrounding environment. Depending on the Copyright © 2002 Marcel Dekker, Inc. regulatory control of gene expression, two types of microbial enzymes are synthesized in waters and sediments: constitutive enzymes, whose synthesis is constant regardless of the presence or absence of the substrate in the environment, and inducible enzymes, whose rates of synthesis are strongly dependent on the presence of their substrates (or substrate derivatives). Many inducible enzymes are synthesized at a low basal rate (i.e., are constitu- tive) in the absence of a substrate. When the substrate is available in the environment, there is a dramatic increase in the production rate of the particular enzyme. Synthesis continues at this amplified rate until the inducer is removed and/or the product of enzy- matic catalysis accumulates (24) and it then returns to the basal rate. Most of the ectoenzymes synthesized by aquatic microorganisms are catabolic en- zymes involved in degradation of polymeric substrates that are not continuously available in the water or sediments. Therefore, the constant synthesis of ectoenzymes in the absence of substrates is unnecessary, because it requires the expenditure of energy that otherwise may be channeled into other useful activities. Since microorganisms have been competing with each other for millions of years, the evolutionary advantages of induction are readily apparent. Most of the ectoenzymes found in freshwaters are inducible, and only a few have a constitutive nature (e.g., some amylases or proteases in bacteria). A. Induction and Repression/Derepression of Ectoenzyme Synthesis The efficient induction of ectoenzymes is more complicated than that of intracellular en- zymes. First, many of the ectoenzyme substrates present in fresh water are polymeric compounds, and they are too large to enter the cell and serve as inducers of synthesis. Second, for an ectoenzyme to be secreted at appropriate rates, the microorganism must be able to monitor the activity of the ectoenzyme outside the cell. We suggest that these problems are overcome by a low constitutive rate of ectoenzyme secretion. If the substrate is present, then low-molecular-weight products accumulate to a certain level, enter the cell, and serve as the inducer (20). When environmental conditions inhibit an ectoenzyme activity (e.g., unsuitable pH, absence of activating cations Mg 2ϩ ,Zn 2ϩ ), the induction of its synthesis does not occur because the product of catalysis is not generated. However, since the microorganisms in freshwater ecosystems are in a complex relationship with a variety of readily utilizable compounds of autochthonous and allochthonous origin, the induction of a particular ectoenzyme by an end product resulting only from degradation of a single polymeric substrate seems to be questionable. Until now, it has appeared that one ectoenzyme may have several inducing compounds (24,37). It is well documented that synthesis of many ectoenzymes produced by aquatic microorganisms is repressed by the end product that accumulates in the cell or in sur- rounding environment. The repression of alkaline phosphatase synthesis by inorganic phosphate (the end product of phosphomonoester hydrolysis) in microalgae and bacteria is probably one of the best-known examples (11,13,38,39). In Lake Plußsee, the specific activity of APase significantly decreased when the ambient orthophosphate concentrations were higher than 0.5 µM (13). APase activity was inversely related to the amount of intracellular phosphorus stored (P st ) in algal cells. When P st constituted less than 10% of the total cellular phosphorus, the algae produced alkaline phosphatase with a high specific activity, and when P st was higher than 15% and the ambient orthophosphate concentrations exceeded 0.6 µM, this activity rapidly decreased. The synthesis of virtually all ectoenzymes in most aquatic microorganisms is re- Copyright © 2002 Marcel Dekker, Inc. Figure 2 Effect of water supplementation with dissolved organic matter extracted from phyto- plankton on growth of bacteria (A) and specific activity of bacterial β-glucosidase (B) and aminopep- tidase (C) in Lake Mikołajskiet. (Chro ´ st, unpublished.) pressed when they are grown on sources of readily utilizable dissolved organic matter (UDOM). This mode of regulation is called catabolic repression. When water samples from eutrophic Lake Mikołajskie were supplemented with dissolved organic matter ex- tracted from phytoplankton (both UDOM and polymeric compounds) the bacterial cell numbers increased markedly during 96 hours of incubation (Fig. 2A). Contrary to that in control samples, supplementation of lake water with phytoplankton organic matter resulted in a significant decrease in the rates of specific activities of bacterial β-glucosidase and aminopeptidase (calculated per bacterial cell) during the first period of bacterial growth (6–48 hours). However, in both of these enzymes, specific ectoactivity began to increase after 48 hours of bacterial growth. In control samples, where bacteria grew solely on naturally present DOM in lake water, the specific activity of these ectoenzymes increased within the incubation period (Figs. 2B, 2C). The repression of ectoenzymes is tightly coupled to the availability of UDOM in Copyright © 2002 Marcel Dekker, Inc. lakewater.Figures2Band2CshowthatectoenzymesynthesisinDOM-enrichedsamples wasnolongerrepressedwhentheconcentrationofthereadilyutilizablelowmolecular- weightmoleculesfellbelowacriticallevel,andpolymericsubstrateshadtobeusedto supportthegrowthandmetabolismofbacteria.Similarinsituobservationsduringphyto- planktonbloomdevelopmentandbreakdownwerereportedforβ-glucosidaseactivityin eutrophicLakePlußsee(24),forβ-glucosidaseandaminopeptidaseactivitiesinmeso- trophicLakeScho ¨ hsee(25),andforlipaseactivityineutrophicLakeMikołajskie(40). Despitethewidespreadoccurrenceofcatabolicrepression,withtheexceptionof thoseforentericbacteria,themoleculardetailsoftherepressionarepoorlyunderstood. Somestudieshaveindicatedthatcyclicadenosinemonophosphate(cAMP),togetherwith itsreceptorprotein,mayplayacentralroleincontrolofcatabolicrepression(41,42). Usingtherepressionstrategyforectoenzymesynthesis,microorganismscanavoidthe wastefulproductionofinducibleenzymes,whicharenotusefulwhentheirgrowthisnot limitedbyUDOM(3,19,24,35). B.InhibitionofActivity Itisimportanttoconsiderthattherepression/derepressionofanectoenzymenotbe equatedtothereversibleinhibitionofactivity.Evenifanectoenzymeissynthesized,its activitymaybeinhibitedbytheaccumulationoftheendproductorbyhighconcentrations ofthesubstrate(19).Twogeneraltypesofreversibleinhibitionareknown:competitive andnoncompetitiveinhibition. Competitiveinhibitionoccurswhenaninhibitingcompoundisstructurallysimilar tothenaturalsubstrateand,bymimicry,bindstotheenzyme.Indoingso,itcompetes withanenzyme’snaturalsubstratefortheactivesubstrate-bindingsite.Thehallmarkof competitiveinhibitionofmanyectoenzymes(e.g.,alkalinephosphatase,β-glucosidase, aminopeptidase)isthatitdecreasestheaffinityofanectoenzyme(anincreaseofthe apparentMichaelisconstantisobserved)forthesubstrateand,therefore,inhibitstheinitial velocityofthereaction(Fig.3)(13,26,37).Competitiveinhibitionisreversibleandcan beovercomebyincreasedsubstrateconcentration,andthereforethemaximumvelocity (V max )ofthereactionisunchanged(Fig.3A). Noncompetitive inhibition generally is characterized as an inhibition of enzymatic activity by compounds that bear no structural relationship to the substrate. Therefore, the inhibition cannot be reversed by increasing the concentration of the substrate. It may be reversed only by removal of the inhibitor. Unlike competitive inhibitors, reversible noncompetitive inhibitors cannot interact at the active site but bind to some other portion of an enzyme-substrate complex. This type of inhibition encompasses a variety of inhibitory mechanisms and is therefore not amenable to a simple description. Noncompetitive inhibi- tion of the activity of exoproteases by Cu 2ϩ ions (43) and inhibition of α-glucosidase, β- glucosidase, N-acetyl-glucosaminidase, and alkaline phosphatase by H 2 S in natural waters have been described (12). C. Environmental Control of the Synthesis and Activity of Ectoenzymes The complex environmental regulation of ectoenzyme synthesis and activity has been demonstrated in studies of bacterial β-glucosidase and aminopeptidase in surface waters of lakes (24,25,37). The results of these studies demonstrated that the ectoenzyme synthe- Copyright © 2002 Marcel Dekker, Inc. Figure 3 Competitive inhibition of β-glucosidase activity by glucose (end product of enzyme reaction) in water samples from eutrophic Lake Mikołajskie. (A) Hyperbolic relationship between enzyme activity and increasing substrate concentrations, (B) Lineweaver-Burk’s linear transforma- tion of the relationship between enzyme activities (1/v) and increasing concentrations of substrate (1/S). (Chro ´ st, unpublished.) Copyright © 2002 Marcel Dekker, Inc. sis and activity were under different control mechanisms, which were dependent on the physical-chemical conditions of the habitat. There is ample evidence for general catabolic repression of ectoenzyme synthesis in bacteria due to readily utilizable carbon sources (24,37,44,45) as well as more specific repression by end products of enzyme catalysis (34,46). However, control of aminopeptidases appears to be distinct and more complex than that of other ectoenzymes. In some bacteria, amino acids, peptides, and/or proteins seem to induce aminopeptidase synthesis (45,47). It is not known specifically how amino- peptidase induction operates, especially since amino acids are reported to act as inducers in some bacteria, rather than acting in their more predictable role as end-product inhibitors. The ability of bacteria living in the euphotic zone of the lakes to produce ecto- enzymes seems to be strongly affected by the availability of the low-molecular-weight, readily utilizable substrates exudated by algae (eg., excreted organic carbon-EOC), which are known to be excellent substrates for bacteria (48–50). Chro ´ st and Rai (25) found that the rates of leucine-amino-peptidase and α-glucosidase production by aquatic bacteria strongly depend on bacterial organic carbon demand. When the amount of EOC fulfilled the bacterial organic carbon requirement, microorganisms did not synthesize enzymes needed for hydrolysis of the polymeric substrates because their utilization was unneces- sary. Moreover, the specific activity of aminopeptidase correlated negatively to the rates of algal EOC. During the active growth of phytoplankton, algal populations excrete into the water a variety of photosynthetic products, including easily assimilable low-molecular-weight substrates (51), which support bacterial growth and metabolism. These substrates inhibit the activity and repress the synthesis of ectoenzymes in bacteria. On the other hand, when low levels of readily available substrates limit bacterial growth and metabolism, bacteria produce ectoenzymes with high specific activity to degrade polymers and other nonlabile substrates. Such a situation occurs in lake water during the breakdown of phytoplankton bloom. Senescent algae liberate, through autolysis of cells, a high amount of polymeric organic compounds (polysaccharides, proteins, organophosphoric esters, nucleic acids, lipids, etc.), which induces synthesis of ectoenzymes. Another mechanism that causes repression cessation of enzyme synthesis is low level of directly utilizable organic com- pounds in the water during bloom breakdown (52). Bacteria living in the profundal zone of the lakes are often substrate-limited (2,53) because the amount of substrate in deep waters depends primarily on the sedimentation rates of the organic matter that is produced in the euphotic zone. There is no direct supply of labile organic compounds exudated by algae. In the profundal zone, sedimentation provides labile monomeric organic compounds that are mostly polymers that are utilized by bacteria. Under such environmental circumstances, bacterial metabolism is strongly dependent on the presence and amount of polymeric substrates and the activity of synthe- sized ectoenzymes that catalyze the release of readily utilizable monomers. Microbial ectoenzymatic activity in natural waters is also strongly dependent on environmental factors, such as temperature, pH, inorganic and organic nutrients, ultraviolet B (UV-B) radiation, and presence of activators and/or inhibitors (3,13,21,54–59). Several studies have shown that ectoenzymes display the highest activities in alkaline waters of pH 7.5 to 8.5 (24,40) or acid waters of pH 4.0 to 5.5 (55). In contrast to the pH response, many ectoenzymes exhibit no obvious adaptation to ambient temperature, because the optimal temperature is often considerably higher than in situ temperature of waters (13,33, 40). The optimal temperatures for alkaline phosphatase and β-glucosidase are unchanged when they are produced by planktonic microorganisms in lake water under different in situ temperatures (13,24). Copyright © 2002 Marcel Dekker, Inc. In light of these aforementioned studies, the environmental regulation of ectoenzyme synthesis and activity is complex and usually no single factor is involved in this process. It is important to realize that environmental regulation of ectoenzymes, induction, synthesis repression, and inhibition are related to concentration, period of exposure, and such factors as temperature, pH, oxygen level, and chemical characteristics of regulatory molecules. The same molecule that is an inducer under one set of circumstances may be a repressor under other environmental conditions, or at different concentrations. IV. ASSAYS OF ECTOENZYME ACTIVITY A. Methods There are significant difficulties in measuring ectoenzyme activities in heterogeneous envi- ronments such as natural waters and soil, which include questions about methodology and data interpretation. For example, should assays be performed according to the well- established principles of enzymology (e.g., excess substrate, optimal pH and temperature, shaking of reaction mixtures) or in situ conditions encountered in an aquatic environment (e.g., limiting and unevenly distributed substrate, suboptimal and fluctuating physical con- ditions, stationary incubation)? How are the optimal assays related to those assays done under more ‘‘realistic’’ conditions? A variety of methods are available for monitoring the enzyme activities when work- ing with microbial cultures or isolated enzymes in biochemical laboratories. However, most classical enzymatic methods cannot be applied directly in aquatic environments. The enzyme amount and activity in natural waters are usually much lower than those measured in cultures or in enzyme extracts, and therefore the classical biochemical methods often are inadequate for measuring low ectoenzyme reaction velocity. Furthermore, the environ- mental conditions of ectoenzyme assays in water samples often are suboptimal (e.g., un- suitable temperature, pH, presence of interfering compounds) and the choice of substrate used to study ectoenzymes of natural microbial assemblages in aquatic environments often is problematic. Depending on the chemical nature of the ectoenzyme substrate, there are three cate- gories of methods for measurement of ectoenzyme activity in aquatic environments: spec- trophotometric, fluorometric, and radioactive. The most commonly used in the past were spectrophotometric methods (60–63). The major disadvantage of spectrophotometric methods is long incubation time necessary for enzyme reactions, which is due to their relatively low sensitivity (micromolar [µM ] to millimolar [mM ] concentrations of the final product of enzyme reaction are required). However, spectrophotometric assays can be used when measuring high enzyme activity in samples, or when working with purified and/or concentrated enzymes. During the last two decades, fluorometric methods have been widely used for en- zyme activity determinations in aquatic environments (3,21,24,33,52,64,65). Fluorometric assays are very sensitive, and they measure the final products of enzymatic reactions in nanomolar (nM) to micromolar (µM ) concentrations. When using a modern spectroflu- orometer to measure enzyme activity in water samples, the incubation time for monitor- ing substrate-enzyme reaction can be shortened to a few minutes. Several authors have applied radiometric methods for enzyme activity determination in aquatic environments Copyright © 2002 Marcel Dekker, Inc. [...]... Schleinsee Lake Depth (m) Pi EHP (µg P-PO 43Ϫ L Ϫ1) Pi/EHP APase inhibition a (%) Constance 2 10 12 20 50 190 1.39 1.80 1.79 6.70 28 .49 59.30 1.64 1.67 1.05 2. 08 0.80 4.53 0.85 1.08 1.70 3 .22 35.61 13.09 77 80 82 91 97 94 1 3 5 7 9 12 1.30 1.80 2. 98 161.51 29 7.60 327 .63 2. 51 2. 20 0.99 0.93 2. 08 1.30 0. 52 0. 82 3.01 173.67 143.07 25 2.03 20 40 85 98 96 99 Schleinsee APase, alkaline phosphatase; Pi, orthophosphate... Edinger Uptake of 32P-phosphoryl from glucose-6-phosphate by plankton in an acid bog lake Int Ver Theor Angew Limnol Verh 24 :21 0 21 3, 1990 92 JT Lehman Release and cycling of nutrients between planktonic algae and herbivores Limnol Oceanogr 25 : 620 –6 32, 1980 93 JT Lehman Nutrient recycling as an interface between algae and grazers in freshwater communities In: WC Kerfood, ed Evolution and Ecology of Zooplankton... proliferation and ectoenzyme synthesis during the assay, changes, which must be prevented They usually are avoided by adding plasmolytic or antiseptic agents to assays, such as toluene or chloroform (10,38,71) However, these agents change the membranes, thereby leading to release Copyright © 20 02 Marcel Dekker, Inc of ecto- and intracellular enzymes In cases in which some enzymes are located intra- and extracellularly... dependence on this function is determined and plotted graphically The results depend essentially on the shape of the hyperbolic curve described by the data, thus making determination of V max and K m difficult (Fig 3A) To obtain these kinetic parameters, the Michaelis-Menten equation often is rearranged to the linear form and V max and K m are obtained from the slope and intercept (Fig 3B) (74) Such graphical... Steiner Zur Kenntnis des Phosphatkreislaufes in Seen Naturwissenschaften 26 : 723 – 724 , 1938 98 W Reichardt, J Overbeck, L Steubing Free dissolved enzymes in lake water Nature 21 6: 1345–1347, 1967 Copyright © 20 02 Marcel Dekker, Inc 99 J Overbeck Early studies on ecto- and extracellular enzymes in aquatic environments In: ´ RJ Chrost, ed Microbial Enzymes in Aquatic Environments New York: Springer-Verlag,... commonly used 7-amino-4-methylcoumarin, 7-amino-4-trifluoromethylcoumarin, and 6-aminoquinoline, are partially protonated at pH Ͻ 5 but fully deprotonated at neutral pH Thus, their fluorescence is not subject to variability due to pH-dependent protonation/deprotonation when assayed near or above physiological pH Substrates derived from water-soluble green fluorophores, fluorescein, and rhodamine, provide significantly... enzyme in fresh waters (3, 117, 124 , 125 ) Orthophosphate ions released by the action of 5′-nase may be either immediately taken up by bacteria producing this enzyme or mixed with the bulk water The fate of the released Pi strongly depends on ambient Pi concentration in the environment and on Pi demand of microplankton In oligotrophic and other Pi-poor waters, release of Pi from 5′-nucleotides by 5′-nase... transformed into bacterial biomass (2, 25), channeled to the microbial loop, and subsequently transferred to higher trophic levels through the grazing food chains The metabolic regulations within the trophic levels and their interspecific relationships are the most important mechanisms steering the flow of organic matter in aquatic food webs Heterotrophic bacteria operating on every level in aquatic food... responsible Copyright © 20 02 Marcel Dekker, Inc for the predominant production of a variety of enzymes involved in the release of nutrients and the transfer of C and energy within the whole lake ecosystem Above all, bacteria are the only single component of aquatic biota that converts the DOM to the particulate phase (bacterial biomass) Moreover, heterotrophic bacteria colonize the detrital POM and, through... 7-hydroxycoumarin (umbelliferone) and the more common 7-hydroxy-4-methylcoumarin (methylumbelliferone) are not fully deprotonated and therefore not fully fluorescent unless the reaction mixture has pH Ͼ 10 (64) Substrates derived from these fluorophores are not often used for continuous measurement of enzymatic activity Products of substrates containing aromatic amines, including the commonly used 7-amino-4-methylcoumarin, . for the enzymes involved in the transformation and degrada- tion of polymeric substrates outside the cell membrane: ectoenzymes (19), extracellular enzymes (20 ), and exoenzymes (21 ). In this chapter, . of these ectoenzymes increased within the incubation period (Figs. 2B, 2C). The repression of ectoenzymes is tightly coupled to the availability of UDOM in Copyright © 20 02 Marcel Dekker, Inc. lakewater.Figures2Band2CshowthatectoenzymesynthesisinDOM-enrichedsamples wasnolongerrepressedwhentheconcentrationofthereadilyutilizablelowmolecular- weightmoleculesfellbelowacriticallevel,andpolymericsubstrateshadtobeusedto supportthegrowthandmetabolismofbacteria.Similarinsituobservationsduringphyto- planktonbloomdevelopmentandbreakdownwerereportedforβ-glucosidaseactivityin eutrophicLakePlußsee (24 ),forβ-glucosidaseandaminopeptidaseactivitiesinmeso- trophicLakeScho ¨ hsee (25 ),andforlipaseactivityineutrophicLakeMikołajskie(40). Despitethewidespreadoccurrenceofcatabolicrepression,withtheexceptionof thoseforentericbacteria,themoleculardetailsoftherepressionarepoorlyunderstood. Somestudieshaveindicatedthatcyclicadenosinemonophosphate(cAMP),togetherwith itsreceptorprotein,mayplayacentralroleincontrolofcatabolicrepression(41, 42) . Usingtherepressionstrategyforectoenzymesynthesis,microorganismscanavoidthe wastefulproductionofinducibleenzymes,whicharenotusefulwhentheirgrowthisnot limitedbyUDOM(3,19 ,24 ,35). B.InhibitionofActivity Itisimportanttoconsiderthattherepression/derepressionofanectoenzymenotbe equatedtothereversibleinhibitionofactivity.Evenifanectoenzymeissynthesized,its activitymaybeinhibitedbytheaccumulationoftheendproductorbyhighconcentrations ofthesubstrate(19).Twogeneraltypesofreversibleinhibitionareknown:competitive andnoncompetitiveinhibition. Competitiveinhibitionoccurswhenaninhibitingcompoundisstructurallysimilar tothenaturalsubstrateand,bymimicry,bindstotheenzyme.Indoingso,itcompetes withanenzyme’snaturalsubstratefortheactivesubstrate-bindingsite.Thehallmarkof competitiveinhibitionofmanyectoenzymes(e.g.,alkalinephosphatase,β-glucosidase, aminopeptidase)isthatitdecreasestheaffinityofanectoenzyme(anincreaseofthe apparentMichaelisconstantisobserved)forthesubstrateand,therefore,inhibitstheinitial velocityofthereaction(Fig.3)(13 ,26 ,37).Competitiveinhibitionisreversibleandcan beovercomebyincreasedsubstrateconcentration,andthereforethemaximumvelocity (V max )ofthereactionisunchanged(Fig.3A). Noncompetitive. (43) and inhibition of α-glucosidase, - glucosidase, N-acetyl-glucosaminidase, and alkaline phosphatase by H 2 S in natural waters have been described ( 12) . C. Environmental Control of the Synthesis