Relationship of metabolite inhibition of growth to flow-of-carbon patterns in nature

Life Sciences Vol . 19, pp . 299-320, 1976 . Printed in the U .S .A .

Pergamon Press

MINIREVIEW RELATIONSHIP OF METABOLITE INHIBITION OF GROWTH TO FLOW-OF-CARBON PATTERNS IN NATURE Robert S . Conrad, John R . Sokatch and Roy A . Jensen Microbiology Department, Universlty Oklahoma, Oklahoma City, Okla ; and Department Biological Sciences, State Universlty New York at Binghamton, Binghamton, New York 13901 . Various genetic diseases arise from biochemical Imbalances that are relatively subtle in the sense that the original mutations are not lethal, that the organism Is most vulnerable to damage during certain phases of rapid development, and that to well-managed cases tt may be possible to avoid damaging effects through the use of appropriate nutritional manipulations . Analogous Imbalances occur in lower organisms . Data obtained with Pseudomonas up tide illustrate that susceptibility to metabolic Imbalance is conditionally dependent upon the nutritional regimen . Stereotsomers of leucine, Isoleuctne and valine, except for L-alloisoleucine, are metabolized as sole sources of carbon and energy by P. up tide . Although the cell yields calculated following utilization of D-leucine and L-leucine were similar, the rate of growth on D-leucine was seven-fold faster than on L-leucine . Slower growth on the L-Isomer is not explained as 2-ketoisocaproate limitation since 2-ketoisocaproate production from L-leucine appears to occur more readily than from D-leucine . Spontaneous mutants were obtained which grew 2-10 times more rapidly than wild type on _L-leucine, L-isoleucine, or L-valine . It is concluded that the true growth potential (rate) of wild type on any of the branched-chain amino acids is masked by a partial, sustained inhibitory effect produced by the cor responding keto acids or their derivative metabolites . Inhibition of growth rate was only found during utilization of branched-chain amino acids as the sole source of carbon and energy, indicating that the metabolite vulnerability is unique to particular flow-of-carbon patterns during growth . The partial and sustained depression of growth rate by branched-chain amino acids in the absence of other carbon sources cannot be attributed to mis-regulation events localized within the biosynthetic pathway . It is concluded that the catabolism of branched-chain amino acids produces a generalized state of metabolic imbalance owing to the existence of abnormally high levels of degradative metabolites such as keto acids or CoenzymeA derivatives . Such compounds could (I) interfere with keto acid (e .g . pyruvate) metabolism, (lt) cause feed-forward inhibition of rate-limiting steps in the pathways of branched-chain amino acid catabolism, (ill) perturb fatty acid composition or disrupt the biochemical integrity of membrane material, or (Iv) react with substrate-ambiguous enzymes, either slowing essential blochemtw 1 299

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reactions to rates that are growth-limiting or producing erroneous products having antimetabolite properties . These effects of branched-chain amino acids in P . up tide may be quite relevant to the molecular events that characterize maple syrup urine disease in man . Metabolite inhibition is probably more caman in nature than is generally appreciated, and an appreciation of the molecular bast's for anomalous Inhibitions of growth in prokaryotic systems should help supply Insight into various molecular diseases in man, many of them yet to be described . In man an ever-expanding literature (i,2) contains descriptions of metabolic disorders that can be traced to mutational alterations of single genes . Such molecular diseases, commonly termed "inborn errors of metabolism" usually equate with a pathological syndrome that 1s triggered by the abnormal accumulation of one or more intracellular metabolites . Intermediary metabolites are ordinarily present at extremely low Intracellular concentrations, and it is not surprising that metabolite accumulation behind an enzyme block might produce metabolic imbalance and hinder normal growth . In multicellular organisms, relaXively subtle primary effects may perturb Interdependent developmental processes, producing an overwhelmingly complex array of pletotrophic effects . Rapidly proliferating tissue (such as In the post-natal brain-growth spurt) may be especially vulnerable to growth inhibition (3,4) . Abnormally high concentrations of an intermediary metabolite could interfere with unrelated enzyme activities of any cell-type In nature, but effects in a multicellular organism may be dramatically amplified by the opportunity for numerous secondary effects since the process of differentiation Involves a wide diversity of metabolic interactions . Hence, an apparent qualitative difference in metabolite sensitivity may appear to distinguish microbial cells from higher eukaryotic cells . This Impression is reinforced by a large molecular biology literature, dominated by Escherichia colt , which contains numerous descriptions of mutants that possess various primary blocks without notable secondary effects . This article reviews evidence to show that microorganisms do in fact exhibit a wide range of metabolite sensitivities . Some microbial groups are especially sensitive to the presence of organic metabolites (e .g ., photoautotrophs and chemolitotrophs) . Even E . toll is susceptible to a number of metabolite-sensitive effects upon growth . Detailed evidence obtained with Pseudomonas Pu~tt~da is presented in this arttele to illustrate that the expression o~ metabolite inhibition may exhibit a conditional dependence upon the flow-of-carbon pattern during metabolism . It seems likely that vulnerability to metabolite Inhibition Is a universal, albeit a variable and sometimes subtle, cellular phenomenon . An understanding of genetic defects such as galactosemia has already benefited greatly through insights derived from analogous charactertsttcs of microbial mutants deficient in galactose-l-phosphate uridyltransferase (5) " For most molecular diseases of man, a manipulative microbial system probably exists that is suitable to serve as an experimental model for the disease . BACKGROUND EVIDENCE FOR METABOLITE INHIBITION IN MICROORGANISMS Inhibition of microbial growth by such cortmon organic metabolites as amino acids Is widespread (6) . Although amino-acid Inhibitions may be especially prevalent In autotrophlc organisms (7), even nutritionally versatile heterotrophs are susceptible . Frequently-cited examples Involve the family of

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branched-chain amino acids . Thus, branched-chain amino acids may cause growth inhibition to Bacillus anthracis (8) . L-Valine inhibits growth In Pseudomonas aeru inosa 9 , and L-valine sensitivity in the K-12 strain of Eschertcha colt 10 is particulârly well known . Some strains of _E . colt 11 and Hydrogenamonas ~. (7) are growth-Inhibited by L-leucine . Saccharomyces cerevisiae may be growth-inhibited by L-Isoleucine, L-valine and a- aminobutyric acid (12) . In man, keto acids dértved from brenched-chain amino acids have been Implicated as the etlologtcal agents responsible (directly or Indirectly) for the Inborn error of metabolism, maple syrup urine disease (13) . Metabolite inhibitions may only be expressed under certain fortuitous condtttons of nutrition, i .e ., the expression of Inhibition may be conditionally Calhoun and Jensen (14) showed that P . dependent upon the growth regimen . ~aeru ~moss exhibited profound variations in susceptibility to growth tnhibition by analogues of aromatic amino acids, depending upon the source of carbon and energy during growth . Vulnerability to inhibition did not correlate with growth rate obtained on various carbon sources . It was concluded that the flow of Intermediates Into the aromatic biosynthetic pathway varied substantially with the metabolism of different carbon sources . Antimetabolite activity with a variety of amino acid analogues was found to be conditionally dependent upon the flow-of-carbon pattern during growth of Serratia marcescens (Calhoun and Jensen, submitted for publication) . In this communlcatton we report the partial, sustained Inhibition of growth by L-Isomers of leucine, valine or Isoleuctne . These Inhibitions are expressed only under a flow-of-carbon pattern where the growth Inhibitor is also the sole source of carbon and energy . Resistance to Inhibition Is readily achieved by mutation . There is considerable support for the generalization that differing nutritional conditions establish an array of flow patterns for intermediary metabolites, differentially stressing (or favoring) particular biochemical pathway sequences . This generalization may be significant (i) In order to develop a full appreciation of various metabolic vulnerabilities and (ii) as an approach that wn be used to stress particular metabolic sequences In order to observe regulatory relationships or to provide selective conditions for the isolation of mutants . MATERIALS AND METHODS Or seisms and rowth conditions . P . up tlda Strain PpG2 (15) (streptomycin res stant, ATCC 232 7 , a leuc ne âuxotroph (H466), and an isoleucine-valine auxotroph (H414), were obtained from Dr . I . C . Gunsalus at the University of Illinois . The auxotrophs were used In cross-streaking procedures designed to detect possible over-production of amino acids to mutants . Stock cultures were maintained on tryptose blood agar base sl$nts and were routinely transferred every month . P . up tide was grown at 30 C in the basal median of Jacobson (16) with carbon sources added at indicâted concentrations . Vigorous aeration was provided during growth by rotary shaking . Growth rates and cellular yields were determined from data obtained with cultures grown In 250 ml side-arm flasks containing 50 ml of medium . The proportionalities between Klétt readings (Klett-Summerson colorimeter, red filter 640-700 nm), optical density et 660 nm and dry weight were correlated by means of standard curves . Inoculum was provided by overnight cultures of cells (0 .5$ glucose minimal salts medium) added at the ratio of 1 ml per 100 ml of fresh medium . Stock solutions of glutamate used as carbon source were titrated to pH 6 .8 with KOH . Isolation of mutants with enhanced abilities to metabolize branched-chain

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amino acids . Spontaneous mutants were selected by growth of wild type in basal medium containing one of the branched-chain amino acids as sole carbon source at 30o C . The cultures were grown to late exponential phase . A 0 .1-m1 volume was trénsferred to 50 ml of 0 .2$ (w/v) glucose medium, and the culture was Incubated until the late exponential phase of growth . Transfer of 0 .1 ml of these cells back to branched-chain amino acid medium during subsequent growth selected for mutants having an enhanced ability to metabolize a given amino acid as a source of carbon . This procedure was repeated three to four times prior to plating the cells on agar containing the selective amino acid . Mutants were readily distinguished from wild type by their larger colony size . Individual mutants (from separate flasks 'to avoid sibling mutants) were purified by three series of single colony Isolations on solid selective medium . Fifty isolates of each resistance phenotype were purified . All . mutants were derived from wild type except the leu/vat double mutant (in which case, valine-resistant mutants were selected from the leucine-resistant parent strain) . Mutants described in this communication carry the following stock denotations : L-leucine-resistant (SCL-1) ; L-isoleucine-resistant (SCI1) ; L-valine-resistant (SCV-1) ; and L-leu/L-vat-résistant (SCLV-1) . Growth~determtnatlons on solid medium . Bacterial lawns were prepared on so id media by spreading 0 .1 ml o glucose-grown cells on media containing 0 .5$ of branched chain amino acid . Solid additives under test as antagonists of branched-chain amino acid Inhibition were placed on the center of lawns on the surface of agar plates . Possible amino acid excretion In resistant mutants was tested by two methods used to detect possible syntropic crossfeeding : transfer of resistant mutants by toothpick to appropriate auxotrophtc lawns previously spread on minimal-glucose agar plates, or by crossstreaking the resistant mutants with auxotrophs on minimal-glucose agar plates . Pre ration of cell-free extracts . Unless otherwise indicated, cultures grown to 0 .5 w v glucose-minimal salts medium were harvested with the use of a Sorvall RC 2-B refrigerated centrifuge at 10,000 x ~ for 15 min . The cells were washed at 4 C in 50 mM K phosphate buffer at pH 7 .5 . Cells suspended (n the same buffer were disrupted by sonic oscillation (Biosonik model Bio-II, power setting 70 for 75 - 90 seconds) or through the use of a French pressure cell . Celluaar debris was removed following centrifugation at 10,000 x ~ for 15 min . a t 4 Ç. These preparations are referred to as crude extracts . Protein concentration was estimated by the method of Lowry _at _al . (17) with bovine serum albumin as a reference standard . Enzyme assays . Four biosynthetic enzymes were assayed under stabl& conditions in crude extracts . Each acttvtty was stable during storage at 0-4 C for at least a week . The acttvtty of threonine deaminase was assayed by fôllowing the appearance of 2-ketobutyrate (18) . The reac~ton was terminated with a hydrazine reagent after 15 min . Incubation at 37 C . The concentrations of 2ketobutyrate was estimated by comparison with a standard curve . Acetohydroxy acid synthetase was assayed by the metho$ of Bauerle et al . (19) . The reaction was terminated after 5 min . a t 37 C by the addition of 0 .1 ml of 12$ H 2 S0 4 , which converts acetolactate, the product of pyruvate condensation, to acetdtn . Acetotn concentration was determined by the procedure of Westerfteld (20) . a -Isopropylmalate (IPM) synthetase was assayed by the procedure of Kohlhaw and Leery (21) . Acetyl-CoA was synthesized as described by Stadtman (22), a method based upon the procedures of Simon and Shemin (23) . Under the assay conditions used, 10 nanomoles of Coenzyme-A corresponded to an optical density increment of 0 .090 . Branched-chain amino acid transaminase was measured by ~he method of Taylor and Jenkins (24) . After 10 min . of incubation at 37 C, the reaction was terminated by hydrazine reagent . Hydrazone concentrations were determined by comparison with a standard curve .

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Growth Inhibition in Biological Syatema

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Amino acid uptake . Cells l ~cttvely growing in minimal-salts medium were used to measure the uptake of C-labeled amino acids . Overnight cultures were inoculated Into 50 ml of fresh medium to 250-m1 sidearm flasks . When turbidtty increased from an initial 10 Klett units to ~~ Klett units, duplicate C-amino acid (approxisamples of 0 .9 ml were added to 0 .1 ml aliquots of mately 0 .25 uC) at the designated concentrations . After two minutes of uptake, the cells were collected on 25 mn Schleicher and Schuell membrane filters (0 .45 u pore size) and washed with 20 ml of the same medium . The filters were placed in scintillation vials and dried under an Infrared heat lamp . A 10-m1 volume of Omnifluor scintillation fluid (New England Nuclear Corporation) was added, and the samples were counted for flue minutes Tn a Beckman CPM liquid scintillation counter . Increases to turbidity were negligible during the uptake interval used . LI id anal ses of cells rown on different carbon sources . Approximately 3 9 wet we ght of cells were obtained from the growth o wild type on DLmixtures of the branched-chain amino acids, glucose, or glutamate . Growth was monitored spectrophotometrically and allowed to reach early stationary phase for all carbon sources . Cells were harvested by centrifugation and washed twice in 50 mM phosphate buffer, pH 7 .5 . Total lipids from pellets of washed cells were extracted and washed according to Folch et al . (25) . The fatty acid composition of total lipids was determined by gâs-lliquid chromatography . Total lipids were transmethylated with boron trtfluoride methanol reagent (Applied Science Laboratories ; State College, Pennsylvania) according to the procedure of Metcalfe and Schnitz (26) . The fatty acid methyl esters were extracted with hexane and purified by thin-layer chromatography . Methyl esters were Identified by comparing their Rf with known standard methyl esters (Applied Science Laboratories) . Thin-layer chromatography was also used to fractionate methyl esters by classes depending upon the degree of unsaturation (27) . In this procedure fatty acids of total extractable lipids were quantitatively determined as their methyl esters by gas-liquid chromatography . The 6 ft ., 0 .5 mm inside diameter column of an Antek 300 chromatograph fitted with a flame detector was packed with 12$ dlethylene glycol succinate on - 80 mesh ABS Anakrome . Cp-~ fatty acid methyl esters were detected at 1707ôC and C 14 -C acids at 3 200 ~ at a carrier gas flow rate of 40 ml/min . The wefficient ~~ variation in récoveries of individual asters from the mixture of six methyl esters (three analyses) was less than 4 .0$ . Total recovery was 95$ and C 8, and C 1 were clearly resolved . Bacterial fatty acids were Identifie 6~ com~p~~ir5g peak retention times and relative retention times to those of standard compounds . Quantitatton of individual components was accomplished by calculating the product of peak height and retention time (28) . Chemicals : Amino acids were obtained from~Sigma Chemical Company . Other chiIs were from Sigma Chemical Company or Calbtochem . All chemicals were of the bast analytical grade commercially available . e METABOLITE INHIBITION IN PSEUDOMONAS PUTIDA Growth on branched-chain amino acids . Each of the branched-chain amino acids can be used by P . Put~tda~ as a sole source of carbon and energy during growth . Cell yields obtained with stereoisomers of branched-chain amino acids are given in Table 1 . Growth on valine (flue carbons) produced a lower cell yield than on leucine (six carbons) or isoleuctne (six carbons), as expected . Although the theoretical ATP yield expected from the complete catabolism of isoleuctne and leucine is similar, the cell yields obtained differed significantly . Comparison of the data obtained with the isoleuctne Isomers

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suggests that one of the four stereoisomers present in commercial DL-Isoleucine is not metabolized . This is undoubtedly L-allo-isoleucinesince the other three enantlomers are utilized (comparing 1 -Înes 2 and 3, Table 1) . The deamlnatioi~ product of L-olio-isoleuclne, 2-keto-3-methylvalerate, is known to be readily utilized by P . ~utida (29) . Therefore, P . up tide is probably unable to convert L-olio-isoleuctne to 2-keto-3-methy3valerate . TABLE 1 Call Yields of P . puttda Grown on Different Substrates

Carbon Source (5~) D-Glucose _D-Isoleucine (with alloy+ L-Isoleucine (olio- free) b DL-Isoleucine (4 Isomers) D-Isoleucine (with alloy L-Isoleucine (alto-free) DL-Leucine D-Leucine L-Leucine DL-Valine D-Valine L-Valine

Cell Yleld a (g x molè 1 )

Ratio dry wt bacteria/wt carbon source

91 .3

o .5t

95 .7 62 .1 94 .3 96 .3 72 .4 65 .1 74 .5 64 .0 55 .6 67 .6

0 .73 0 .47 0 .72 0 .74 0 .55 0 .50 0 .57 0 .55 0 .48 0 .58

a Cell yields obtained are expressed as dry weight of bacteria produced per mole of substrate . b Equlmolar amounts each Isomer . Surprisingly variable growth rates were observed during metabolism of the Individual branched-chain amino acids as sole source of carbon (Table 2) . A seven-fold faster growth rate on D-leucine than on L-leucine was particularly striking . Growth on D-leuctné was about twice âs fast as on isomers of Isoleucine and about four times as fast as on isomers of valine . The catabolism of leucine isomers In P . -putide is Identical following the first enzyme step of degradation (30) . D-LeurTne and L-leucine are each deaminated to 2-ketoisocaproate by the câtalytic actions of D-amino acid dehydrogenase and branched-chain amino acid transaminase, respectively (31) . Hence, it appeared that the difference In growth rate would most likely be explained either by differential efficiency of transport for leucine isomers or to differences in rate of enzymatic deamlnation of the two Isomers . Slower growth on L-leucine than on D-leucine would be consistent with a poor transport capability for L-leucine l ~elative to D-leucine . However, a sertes of transport experiments with C-labelled isomers established a qualitatively more rapid transport of L-Leucine than of D-leucine (even when transport was assayed in cells previously conditionedto growth on the D-Isomer, in the event of inductble transport proteins) . For example, at a concentration of 0 .01 mM, the uptake velocity for L-leucine was 2 .0 nanamoles per mg dry weight per min campa red to 0 .15 nanamolé per mg dry weight per

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min for D-leucine . Although growth medium initially contained 25mM of external âmino acid, control experiments established that 0 .01 mM is a saturating concentration in short-term experiments .

TABLE 2 Growth Rates of P . putida on Branched-chain Amino Acids Doubling Carbon source s time (hours) b L-Glutamate D-Glucose D-Leucine L-Leucine D-Isoleuctne L-Isoleucine D-Valine L-Valine

1 .0 1 .2 2 .8 20 .4 5 .1 5 .5 11 .4 10 .4

a Carbon source concentrations were 25mM, an amount not growth-limiting with respect to yield . L-Isoleucine was silo-free ; D-isoleucine was not . b Doubling time is calculated from the slope of semi-log plots of cultures growing exponentially at 30oC . In the case of long doubling times, care was required to recognize the takeover of same cultures by faster-growing mutants . For this reason, the doubling-time values obtained for L-leucine, D-valine and _L-valine may tend to be smaller, if anything, than the true values . A second possible explanation for the faster growth rate sustained on the DIsamer of leucine would be that branched-chain amino acid transaminase Is the rate-limiting reaction to the degradation of L-leucine, whereas D-amino acid dehydrogenase is not rate-limiting in the degradation of D-leucine . This explanation seems unlikely since the specific activity ofbranched-chain amino acid transaminase is at least 30 - fold greater than that of D-amino acid dehydrogenase, even following maximal induction of the dehydrogenâse (31,32) . Data illustrating these specific activity differences are shown in Table 3 . Finally, if slow growth on L-leuctne were the result of 2-ketolsocaproate limitation as a consequenceôf a relatively slow transport rate for Lleucine or a relatively slow deamtnation of L-leucine, then direct addition of 2-ketoisocaproate to L-leucine-growing cultures would be expected to at least partially stimulaté growth . No such stimulatory effect was found . Metabolite-resistant mutants . The latter results indicate that, tf anything, exogenous L-leucine probably is converted to endogenous 2-ketoisocaproate more readily than Is the D-Isomer . Hence, rather than being growth-limiting as originally supposed, accumulated 2-ketoisocaproate or some derivative metabolite probably Inhibits growth . If the growth rate on L-leucine does reflect a partial inhibition, then it is likely that resistant mutants could be Isolated that would exhibit faster growth . Spontaneous mutants were indeed selected as faster-growing colonies arising at high frequency from

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Growth Inhibition in Biological Syatema TABLE 3

Effects of~Carbon Source Upon Enzymic Production of Branched chain Keto Acids Specific Activity (nmol/min/mg protein) Carbon source

Enzyme s substrate

D-Amino acid b Branched-chain Dehydrogenase aminotransferase

_DL-Leucine

Leucine Isoleuctne Valine

6.0 5.7 7.1

558 364 198

_DL-Isoleuctne

Leucine Isoleuctne Valine

8.6 8 .8 12 .5

408 487 -

_DL-Valine

Leucine Isoleuctne Valine

7.3

6 .0

20 .0

572 -

_D-Glucose

Leucine Isoleuctne Valine

0 .0 0 .0 0 .0

554 340 224

°Enzyme activities of D-amino acid dehydrogenase and branched-chain aminotransferase were specific, respectively, for the D- and L-isomers . D-Amino acid dehydrogenase utilized bath D-lsoleucine andD-allo-lsoleucine with bnegltgible differences in observed specific activity . Assayed as described by Martin et al (30) . TABLE 4 Mutant Changes in Pattern of Carbon Source Utilization Doubling time (hours) b Carbons Source

Wild Type

Leucine Resistant

lsoleucine Resistant

D-Glucose D-Leucine L-lsoleucine L-Valine L-Leuc i ne

1 .2 2 .8 5 .5 10 .4 20 .4

1 .1 2.7 2.4 ~ 10 .4 1 . 8 ~(`

1 .0 2 .Of 1 .9f 1 .8~ 5 . 9t"

bThe concentration of each carbon source was 25 mM .

Valine Resistant

1 .2 ~r4 .3 ~13 .5 2 .1~ {~28 . 6

Leu/val e Resistant

1 .0 2 .8 2 .7~ 3 .2 ~ 1 .8 {~

Arrows Indicate mutation to increased growth ratë (~) or decreased growth rate (~) c Double mutant . Selection for valine resistance was made beginning with the L-leucine resistant mutant .

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Growth Inhibition is Biological Systems

the background of wild-type lawns spread on agar plates containing Lleucine, L-isoleucine or L-valine as the sole source of carbon . The altered phenotypic pattern of growth on branched-chain amino acids was characterized for several types of resistant mutants . Table 4 shows that P . up ttd~ possesses the intrinsic potential to grow on each L-isomer with â doubling time of less than two hours . An Interrelated basis for the indivtdual~amino acid toxicitles is suggested by the pletotrophtc effects of each mutation (Table 4) . For example, mutat .lon to a dramatic increase In growth rate on L-leucine simultaneously improved the rate of L-tsoleucine utilization . L-Isoleucine resistance led to an Increased growth rate on all three aminô acids . On the other hand, L-valine resistance led to a concommitant Increase In sensitivity to growth inhibition by either L-leucine or L-isoleucine . Growth rates on L-leucine and L-isoleucine covaried in parâllel for each of the mutants shown, an observation that may imply an antagonistic relationship between valine and the isoleucineleuclne pair . The double mutant shown at the right in Table 4 (leu/val resistant) was obtained by selecting for L-valine resistance in a genetic background of previous mutation to L-leucine resistance . This doublemutant strain grows batter than wild type on each branched-chain amino acid . Growth rates of mutants on D-Isomers did not differ from those of wild type by a factor of more thantwo . Fifty mutants representing each of the four classes shown in Table 4 were purified . Each class appeared quite homogeneous within the qualitative limitations of observing colony size on solid media containing various branched-chain amino acids . Exceptionally, when selection was on Lvaltne, two colony types emerged . A large-colony type (72$ of resistant clones) had the L-valine resistant phenotype similar to that in Table 4 . A smaller-colonytype (28$ of resistant clones) was quite similar to the double mutant (leu/val resistant, Table 4), raising the possibility that this (small colony) mutant type may also contain two mmutatlons . Further genetic studies are required to resolve this question . TABLE 5 Growth of P . utida Wtld Type on Different Combinations ôf Branched chain Amino Acids

Carbon source (5 mM)

Addition (0 .2 mM)

Doubling time (hours) WildLeuclnetype resistant

_L-Leucine

0 L-Valine L-Isoleucine

20 .4 10 .8 22 .8

1 .8 2 .5 1 .8

_L-Valine

0 L-Isoleucine L-Leucine

10 .4 6 .0 9 .0

10 .4 3 .4 7 .4

_L-Isoleucine

0 L-Valine L-Leucine

5 .5 4 .3 9 .2

2 .5 2 .5 3 .4

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Gro+~th Inhibitioa is Biological Systeme

Inhibition of growth to Ante onistic effects of amino and keto gelds . wild type dur ng utilization o a given amino acid is only partially reversed by the addition of the remaining branched-chain amino acids and pantothenate . Growth rate did vary markedly with the addition of same branched-chain amino acid combinations (present at concentrations 25fold lower than the eoneentratlon of the maJor carbon source) . Again an apparent relationship of antagonism for the Isoleucine-leucine pair with Thus, data obtained from wild type (Table 5) valine seems . to exist. show that either L-Isoleucine or L-leucine Increases growth rates on Lvaline, while L-vâltne Increases growth rate on either L-isoleuctne L=leucine. L-Îsoleueine decreases the growth rate on L-leucine, while L-leucine décreases the growth rate obtained on L-Isoleucine . Results obtained with the leueine-resistant mutant are also given in Table 5 . All of the common amino acids were tested, and some were found to produce a more modest antagonism of Inhibition . These effects were probably Indirect, e .g ., Interference with transport, or metabolism of the amino acids with a subsequent alteration of the overall physiological state.

or

Branched-chain amino acid intermediates were tested for qualitatively obvious growth effects on solid media . The keto analogues of isoleuctne, valine and leucine (2-keto-3-methylvalerate, 2-keto-isovalerate, and 2ketoisocaproate, respectively) did exhibit some effects upon growth in wild type and mutants (Table 6) . TABLE 6 Ca rbon Source 0.3$ (w/v)

L-leucine

L-Isoleucine

L-Valine

Effect of Branched-chain Keto Acids Upon Growth w h sea Wlld Leucine- Isoleucine- Valine Leu valb Addition R* R* R* R* type Pyruvate 2-Ketobutyrate 2-Keto-3-methylvalerate 2-IGetoisovalerate 2-Ketoisocaproate

S I

S I

S I

S I

S I

S -

S

S S

S S

S S

Pyruvate 2-Ketobutyrate 2-Keto-3-methylvalerate 2-Ketotsovalerate 2-Ketoisocaproate

S I

S I

S I

S 1

S 1

Pyruvate 2-Ketobutyrate 2-Keto-3-methylvalerate 2-Ketolsovalerate 2-Ketoisocaproate

S

S

S

S

S

S -

I I

S

Of other compoundstestéd -â Inobutyrate tended to mim c the e acts o 2ketobutyrate, although inhibition ôf growth was usually weaker and often variable . Pantothenate, a derivative of 2-ketolsovalerate, did not influ$nce growth inhibition by any ôf the branched-chain amino acids . Symbols : R*, Resistance ; S, stimulation of growth ; I, inhibition of growth ; band -, no effect upon growth . One mg of keto acid was added to the center of an agar plate previously spread with a confluent lawn of the strain indicated .

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Growth Inhibition in Biological 3ysteme

2-Ketobutyrate Inhibited Pyruvate stimulated growth in all cases . growth when L-leucine or L-tsoleucine was the carbon source, but not Hence, 2-ketobutyrate probably when L-valtné was the carbon source . acts primarily as a valine antagonist . Since P . utida does not utilize threonine as a so Fe source of carbon and energy, It occurred to us that this might reflect the toxicity of 2-ketobutyrate produced from threonine . However, we did not Isolate 2-ketobutyrate-resistant mutants In an attempt to select them on threonine-containing media . Secondly, none of the resistant mutants were resistant to exogenous 2-ketobutyrate (Table 6) . Neither threonine nor 2-ketobutyrate Inhibit growth In the presence of other carbon sources such as citrate or glucose . In wild type the stimulatory effects of keto acids upon growth during primary metabolism of an amino acid (Table 6) may reflect the stimulatory ability of the corresponding amino acid (Table 5) . Thus stimulation of growth on L-leucine or L-valine by 2-ketoisovalerate or 2-keto3-methylvalerate, respectively Table 6) is paralleled by growth-stimulatory effects of L-valine and L-tsoleucine (Table 5) . It is apparent that many relationships are changed in various mutants, but no clear interpretations can be drawn until detailed enzymologteal studies are done with the mutants . Enz reacttvit with substrate anal ues . The reactivity of a number o enzymes with structurally re ated keto acids was considered as a possible mechanism of Inhibition . Acetohydroxy acid synthetase possesses a natural substrate ambiguity, condensing two pyruvate molecules to form a valine precursor or one molecule each of pyruvate and 2-ketobutyrate to form an Isoleuctne precursor . Pyruvate and 2-ketobutyrate (structural analogues) may compete for a cortmon substrate site, as Is consistent with the 52$ inhibition by 2-ketobutyrate (Table 7) of the pyruvate condensation assay . TABLE 7 Inhibitors of Acetohydroxyacid Synthetase

Addition lone L-Valine L-Isoleuctne L-Leucine BC-aa mixture

-

2-Ketobutyrate 2-Ketolsocaproate 2-Ketolsovalerate 2-Keto-3-methylvalerate

Concentration mM 5 .0 5 .0 5 .0 S .O b 10 .0 10 .0 10 .0 10 .0

Specific activttya _346 86 132 346 85

$ Inhibition -

166 343 318 354

b Speeifie activity is expressed as nanomoles per mIn per mg protein . Each amino acid present at 5 mM .

Hence, extreme perturbation of the intracellular ratio of pyruvate :2ketobutyrate may disrupt some optimal ratio of tsoleucine : valine formation . This is consistent with the growth Inhibitory action of 2ketobutyrate .

75 62 0 75 52 1 7 0

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An additional possible target of keto acid Imbalance is the final enzyme reaction of the common isoleucine-valine sequence . This reaction Is catalyzed~by branched-chain amino acid transaminase, an enzyme which is ambiguously reactive with the keto analogues of L-leucine, L-valine and L-isoleucine . Tha specific activities measured Tn P . uttdâ for b~anchedchatn amino acid transaminase in glucose-grown cells at saturating concentrations of substrate were 554, 340 and 224 nanamoles per min per mg protein with _L-leucine, _L-isoleucine and _L-valine, respectively (Table 3) " A most striking instance of substrate ambiguity was seen with a-IPM synthetase (Table 8) . TABLE 8 Substrata Specificity of

:a-IPM Synthetase

Substrate a

Relative Activity b

2-Ketotsovalerate 2-Ketobutyrate Pyruvate 2-Ketolsocaproate 2-IGetog 1 utarate 2-Keto-3-methylvalerate

100 109 80 15 0 .4 0

aAl1 Keto acids were used at 20 mM concentration except for 2-ketoisovalerate b (2 mM) . A reTatlve activity of 100 corresponds to a specific activity of 4 .1 nanomoles per min per mg protein . TABLE 9 L-Leuclne Inhibition of a-IPM Synthetase with Different Substrates Specific activity a

Substrate (2mM)

Minus Leuclne

Plus 1mM Leucine

$ Inhibition

2-Ketoisovalerate 2-Ketobutyrate Pyruvate 2 Ketotsocaproate

4 .3 3 .5 3 .2 0 .9

1 .4 1 .7 0 .1 0 .4

67 51 97 56

a

Specific activity is expressed as nanomoles par min per mg protein .

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The enzyme condenses acetyl-CoA with 2-ketobutyrate and pyruvate (substrate analogues) nearly as well as with 2-ketoisovalerate (see Table 9 for comparisons of Identical substrate concentrations) . 2-ketoisocaproate 2and 2-ketoglutarate are utilized less efficiently as substrates . Keto-3-methylvalerate did not exhibit detectable substrate reactivity . Mts-uttltzatlon of such substrates as pyruvate, 2-ketobutyrate, and 2ketotsocaproate results to the formation of 2-methylmalate, 2-ethylmalate and isobutylmalate, respectively (33) . The differential susceptibility of a-IPM synthetase to inhibition by L-leucine depending upon the substrate utilized (Table 9) may be signtfTcant in assessing the _In vivo implications of substrate ambiguity . Re ression control of enz mes in the bios nthetic athwa . The levels o several biosynthetic enzymes were found to vary in response to the presence of amino and keto acids during growth . L-Glutamate was used as the primary carbon source in order to obtain unifôrm cultures for the preparation of extracts yielding the enzyme data given in Table 10 . TABLE 10 Effects of Amino and Keto Acids Upon Levels of Biosynthetic Enzymes Specific activity b

Additive to growth medluma None` L-Valtne d L-Isoleuc~ne d L-Leucine BC-aa mixture° 2-Ketobutyrate (2KB) 2KB + L-isoleuclne 2KB + L-leuctne 2KB + L-valine 2KB + BC-aa mixture L-Threonine 2-Aminobutyrate

a-IPM synthetase

Acetohydroxy acid synthetase

2 .7 23 .1 3 .8 1 .0 0 .9 28 .3 36 .3 15 .7 12 .6 4 .7 10 .0 16 .1

101 126 165 129 88 352 531 226 127 53 114 380

Threonine deamtnase 109 325 107 161 116 124 228 250 133 145 131 105

a The growth medium contained 25 mM glutamate as the carbon source plus 5 mM of each additive indicated . Cultures for extract preparation were harvested to the late exponential phase of growth . b Spectftc activities are expressed as nanomoles per min per mg of protein . Branched-chain amino acid transamtnase activity was also measured and found to be expressed constitutively at a specific activity of about 500 nanomoles per min per mg of protein when leucine was the amino donor . c Slmilar results were obtained when 25 mM glucose was the carbon source . d Stmtlar specific activities were obtained when this amino acid was the sole source of carbon and energy . eMlxture of L-leucine, L-valine and L-Isoteuctne .

312

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Growth Inhibition in Biological Syatema O-~

W~wlr/mrl

FIG . 1 When glutamate was present as the source of carbon, L-valine produced an abrupt, transient inhibition of growth persisting for about two hours (Fig . 1) An exponentially growing culture utilizing 25mM glutamate as the sole source of carbon and energy was diluted to give the cell mass shown at zero time on the abscissa scale . Continued exponential growth was monitored for four hours, at which time (arrow) L-valine was added to a final concentration of 5 mM . Turbidity measurements were continued for 6 hours . Samples for énzyme assays were collected in 50-m1 volumes, sedtmented by centrifugation, and frozen at -80 C until ready for extract preparation . The Interval of inhibition correlates with an increased (3-fold) synthesis of threonine deamtnase . Table 10 shows that a-IPM synthetase also Increases by a factor of about nine . These results suggest that transient inhibition by L-valine Is related to starvation for L-leucine and L-isoleucine, a conclusion reinforced by the finding thattransient InhTbttion by Lvaline Is completely reversed in the presence of L-leucine and L-isoleucine . Transient Inhibition by L-valine was not observedwhen glucose replaced glutamate as the carbon source . Neither L-leucine nor L-isoleucine supplementation of glutamate-growing cultûres produced transient inhibitory affects upon growth or dramatic effects upon enzyme levels . 2-Ketobutyrate inhibits growth (not transient) and causes derepresslon of a-IPM synthetase (10-fold) and acetohydroxy acid synthetase (3-fold) .

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Growth Inhibition in Biological Syetema

313

Conditions that promote growth are accompanied by enzyme derepresston (Table 10) . Inhibition of growth by 2-ketobutyrate is accentuated by Lisoleucine and completely reversed by L-leucine plus _L-valine or by Lvaline alone (L-leucine can be derivedfrom L-valine) Enzyme derepression by 2-ketobutyrâte Is accentuated in the presénce of L-isoleucine, reversed completely by L-tsoleucine plus L-leucine, and partlelly reversed by either L-valine (best) or L-leucine . It is not surprising that 2-amtnobutyraté resembles 2-Ketobûtyrate as an inhibitor of growth . The enzyme levels found In the presence of threonine indicate that exogenous threonine, if utilized at all, is not converted to high Internal levels of 2ketobutyrate . Mis-re ulatton of enz activities In the branched-chain athwa 1 The branched-chain am no ac ds comer se a amt y o structura ana ogues . The possibility was considered that mis-regulation of a regulatory enzyme within the biosynthetic branched-chain pathway by an inappropriate amino acid might cause a slowed growth rate during utilization of carbon source levels of amino acid . Table 11 reveals that a-IPM synthetase activity is indeed inhibited significantly by L-isoleucine In addition to the expected feedback Inhibition by L-leucine . TABLE 11 Inhibitors of a-IPM Synthetase Concentration mM

Specific activitya

None _L-Leucine

2 .0 1 .0 0 .4

8 .9 0 .6 3 .5 6 .6

93 60 26

_L-Isoleucine

1 .0 0 .5

4 .7 6 .1

48 32

D-Leucine

1 .0

8 .2

9

L-Valine

1 .0

9 .2

0

Addition

$ Inhibition

Specific activity is expressed as nanomo es par m n per mg prote n . Secondly, L-leucine Inhibits threontne deamtnase (Table 12) in addition tô the expécted feedback Inhibition by,L-isoleucine . Although both a-IPM synthntase and threonine deaminase are potentially vulnerable to mtsregulatlon effects, acetohydroxy acid syntnetase activity Is only inhibited (Table 7) by L-valtne or L-Isoleucine, a not unexpected regulatory pattern . Altered fatty acid synthesis . The metabolism of branched-chain amino acids is potentially capable of influencing fatty acid synthesis since the utilization of a given branched-chain amino acid Inevitably alters the intracellular proportions of possible lipid intermediates (acylCoenzyme A derivatives) . For example, the catabolism of both valine and isoleucine yields proptonyl-CoA, which may substitute for acetyl-CoA as a primer moiety, thereby producing an odd-chain fatty acid . It seamed possible that retardation of growth might reflect the perturbation of fatty acid composition .

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The chloroform-methanol extractable lipids of wild-type and mutant cells grown on various carbon sources (0 .5$ D-glucose, L-glutamate, and the branched-chain amino acids) were analyzed by gas-liquid chromatography . The fatty acid pattern of wild type varied appreciably following growth on different carbon sources, e .g ., the appearance of peaks corresponding to C15 on valine- or Isoleucine-grown cells that were not found In glutamate or glucose medium . The fatty acid profiles of mutant types described to Table 4 all differed significantly from that of wild type following growth in a given medium . However, no patterns were discerned that might implicate causal relationships of deranged fatty acid synthesis and branched-chain amino acid toxicity . (For example, one might expect a given fatty acid entity to be prominent following growth of wild-type on L-leucine but not following growth on glutamate, and to be prominent in the L-valine-resistant mutant (Table 4) grown on L-leucine but not In the L-leûclne resistant mutant .) We conclude that mutant differences to fatty âcid composition probably reflect affects rather than causes of altered branched-chain amino acid catabolism . TABLE 12 Inhibitors of Threonine Deamtnase

Addition

Concentration mM

Specific activity a

$ Inhibition

_L-Leucine

10 .0 5 .0 3 .5 2 .0 1 .0 O

3 37 72 96 131 125

9$ 72 46 28 0 0

_L-Vailne

10 .0 5 .0 3 .5 0

149 162 167 159

6 0 0 0

5 2 47 111 134 1513

98 99 70 30 15 0

L-Isoleuctne

5 .0 1 .0 0 .1 0 .04 0 .02 O

- Specs tc activity is expressed as nanamoles per min per mg protein . MOLECULAR BASIS FOR GROWTH INHIBITION The conditional nature of rowth Inhibition b branched-chain amino acids . Branched-chain amino acids restrict the growt rate o . ut da only under conditions where the amino acids are essential ifgrows to occur at all (i .e ., when utilized as sole source of carbon and energy) . Consequently, the intrinsic potential of these amino acids to support a more rapid rate of growth Is not readily apparent In wild type . _A rp lori the observation that mutants can grow with faster doubling times on branched-chain amino acids is consistent with either of two entirely

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Growth Inhibition is Biological Systems

different explanations : (i) improvement of some rate-limiting step of carbon source degradation, or (ii) resistance to some endogenous growthinhtbitory agent . An important clue bearing on a choice between these possibilities is offered by the seven-fold difference in growth rate that is observed in wild type, depending upon which isomer of leucine is used as the carbon source . The catabolic pathways for D- and _L-leucine are identical following deamination to 2-ketolsocaproate . If explanation (t) above explains the batter growth on D-leucine (doubling time, 2 .8 hours) than on L-leucine (doubling time, 20.4 hours), then only differences to the two~atalytic events leading to 2-ketoisocaproate could account for different growth rates . Hence, In accord with explanation (i) one might expect the transport of D-leucine to be more efficient than that of Lleucine, or the dehydrogenase-mediated conversion of D-leucine to 2ketoisocaproate to be more efficient than the transaminase-mediated conversion of L-leucine to 2-ketoTsocaproate . Exactly the opposite trends were observed, indicating that both the transport and deamination rates for L-leucine exceed those for D-leucine . Thus, 2-ketoisocaproate rather than being limiting to growth as su Bested by (i)] must be Inhibitory to growth [ as suggested by (tt) ~, either directly or through a metabolite derived from 2-ketolsocaproate . Presumably, rapid growth on D-leucine in wild-type P . utida reflects a near-optimum balance In théformation and uttllzatTon~ketotsocaproate . The plefotrophic properties of mutants also suggest that a state of metabolic Imbalance may provide the basis for growth Inhibition during degradation of Ltsomers . Thus, mutants selected for Improved growth rate on a glvén amino acid inevitably displayed perturbations of growth rata on other branched-chain amino acids . A rather large number of possibilities that might account for the exact nature of the postulated metabolic imbalance are considered in the following sections .

T

Feed-forward inhibition of carbon source catabolism ? Catabolism of branched-chain amino acids involves numerous Coenzyme A derivatives . For example, oxidation of valine results in the formation of isobutyrylCoA, methylacrylyl-CoA, 3-hydroxyisobutyryl-CoA, propionyl-CoA and methylmalonyl-CoA . Substantial accumulation of a particular Intermediate might Interfere with subsequent reactions of branched-chain amino acid oxidation . Since some of the catabolic enzymes In P . up tide are constitutive while others are Inducible by branched-chainâmino acids (32), unbalanced levels of pathway intermediates could arise through such a lack of coordinacy .i n enzyme Induction . For example, although L-valine induces the formation of branched-chain keto acid dehydrogenasé (which forms isobutyryl-CoA), the level of branched-chain acyl-CoA dehydrogenase (which degrades isobutyryl-CoA) remains unchanged . Perhaps an increased accumulation of Isobutyryl-CoA may inhibit one or more of the subsequent reactions, thereby slowing the rate of methylmalonyl-CoA formation and hence the rate of growth . Isobutyryl-CoA may accumulate to a lesser extent during growth on D-valine owing to the relatively slow rate of 2-ketoisovalerate formation by D-amino acid dehydrogenase . It will be Interesting to determine whether any of the various mutants are altered in enzymes of branched-chain amino acid catabolism . The use of common enzymes (e .g ., branched-chain keto acid dehydrogenase) to catalyze analogous reactions in the degradation sequences of leucine, tsoleuclne and valine (32) are consistent with the plelotrophtc effects In mutants whereby an improved growth rate on one substrate results in a changed growth rata on another substrate . Keto acid imbalance .

Some unique characteristics of the biosynthetic

316

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Growth Inhibition in Biological Systeme

pathway for branched-chain amino acids are highlighted in Fig . 2 which is drawn to emphasize the number of the precursor Interrtledlates that are keto acid structures . Catabolism of each amino acid shown results initially Tn ketoleucine, ketovaline, or ketoisoleucine formation . Keto acids have often been reported to Inhibit growth of prokaryotic organisms (7,34) . Among eukaryotic organisms, tha ketoacidurlas of man such as maple syrup urine disease and phenylketonurta are especially noteworthy Inspection of Ftg . 2 shows that the keto acids within the branched(35) " chain pathway comprise a group of analogue structures . Massive catabolism of one amino ac(d and the rapid production of the keto derivative of that amino acid may generate a marked keto acid imbalance . Alteration of the normal ratio of keto acids may perturb certain essential cellular processes, perhaps In analogy to the well-known inhibitory effects upon growth of high sugar-phosphate concentrations under conditions of unbalanced carbohydrate catabolism (36) . FIG . 2 ÇH a

KETOLEUCINE LEUCINE ~1

F1-CFia

R-CFIa -CFF` ~ ~a

Fl-Ç~FI=OH

I"

__

~a KETOPANTOATE __

F~IRWATE

KETOVALINE

KETOBUTYFIATE

KETOIaOLEUCINE ~a CHa~FIa

~:~a

~"

L-VALINE L-IaOLEUCINE

Keto acids within the pathway of branched-chain amino acid biosynthesis . Ketoleucine, ketovaline, and ketoisoleucine are 2-ketolsocaproate, 2-ketolsovalerate, and 2- koto- 3-methylvalerate, respectively . R denotes the structure HOOC-Ç~O . Enzyme [1] is threonlne deaminase, [2] is acetohydroxy acid syntj~~tase, [5] and [7] Is branched-chain amino acid transamtnase, and [6] is a-IPM synthétase . Ketopantoate is a precursor of pantothenic acid . Abnormally high Intracellular concentrations of keto acids may inhibit growth ear se . Possible mechanisms for keto acid inhibition of growth are diverse . Ananalous inhibitions of essential enzyme activities may occur . For example, pyruvate inhibits growth through inhibition of isocttrate lyase In Arthrobacter (37) . The keto acid counterparts of leucine, lsoleucine and va ne are lipophlltc compounds that are likely to orient to membrane material . Several studies (38,39) have shown that lipophilic acids inhibit microbial growth through limitation of organic and Inorganic nutrients, apparently by uncoupling the electron transport system from substrate transport and oxidative phosphorylatlon . We also considered the possibility that fatty acid composition m(ght be disrupted In a way that could retard growth rate . In rat brain keto acids inhibit fatty acid synthesis (13) and lipid metabolism (40) . An Increase In lipid content of mouse fibroblasts was found after exposure to 2-ketoisocaproate, probably the consequence of new lipid synthesis from 2-ketolsocaproate w rbon (41) . Isobutyrate derived from L-valine is substantially Incorporated into iso-branched fatty acids mallmallan

in

Vol . 19, No . 3

Groorth Inhibition in Biological Systems

31 7

In Tatrah na riformls Isovalerate produces surface lipids (42) . growth inhibition, at the sa~incr~y a factor of four the fraction of 1so-derivatives comprising total fatty acid content of polar lipids (43) . It was suggested that growth inhibition is the consequence of derangement of membrane fluidity owing to perturbation of fatty acid composition to polar lipids . We did indeed find that fatty acid composition changed markedly in P . up tide under different growth and carbon source conditions . Many ofour mutants exhibited fatty acid compositions However these differing from those of wild type In a given medium . variations were probably indirect effects of the mutational changes since no definitive features of the various fatty acid profiles could be equated to growth inhibition . Substrate ambt uit . The various keto acid-utilizing enzymes of branchedchain am no acid biosynthesis (Fig . 2) may be especially vulnerable to keto acid Imbalance . Most of these enzymes possess broad specificitles that allow reactivity with a series of related keto acids . This is dramatically Illustrated by . a-IPM synthetase of P . up tide which condenses acetyl-CoA with at least three keto acids to additiôn to 2-ketolsovalerate . A low specificIty of leucine pathway enzymes (33,44), especially a-IPM synthetase, seems to be coamon In microorganisms Including Salmonella typhimurtun (21), P . aeruginosa (33), Neuros ra crassa (45 , S . cerevtstae (46), ând H dro anemones H1 . -In N, crasse the V ~o a-IPM synthetase or 2-ketobutyrate is actually greater than~~r 2-ketolsovalerate although the K for 2-ketolsovalerate Is several orders smaller than for 2-ketobutyrate . It is noteworthy that significant reactivity with 2-ketobutyrate would ba expected in vtvo under conditions of keto acid imbalance where 2-ketolsovalératefs limiting and 2ketobutyrate accumulates . The broad specificity of branched-chain amino acid transamtnase in P . ~aeru ~loose has been reported (48) . An inhibitory effect may be ampli lewd l n vtvo since the erroneous product may interfere with the succeéding enzyme reaction of the sequence . The enzymes of isoleuctne and valine synthesis including both branched-chain amino acid transamtnase and a-acetohydroxy acid synthetase exhibit a natural substrate ambiguity, to which a parallel series of analogue structures is formed . Balanced Intracellular ratios of the competing substrates for these multlfunctlonal enzymes is probably important for optimal pathway function . Intracellular accumulation of 2-ketobutyrate might result In transamtnatlon to 2-amlnobutyrate, an amino acid known as a general Inhibitor of bacterial growth (49) . Aberrant transamination of ketopantoate would tend to inhibit Coenzyme-A synthesis . We recently found (50) that to N . crassa under conditions in which prephenate (an aromatic keto acid) âccumulates, a non-specific transamtnase converts it to substantial yields of pretyrosine (a compound not normally formed in detectable amounts) . Possible mis-re ulation of branched-chain amino acid bios nthesis7 A number o precedents exist for internal mis-regulation of complex pathways . Thus, exogenous L-valine Inhibits the growth of E-toll K-12 as a consequence of Inhibitiôn of L-isoleucine biosynthesis 10 . In P . aeru loose valine does not inhibit growth to a glucose-containingmedium 9 . Ha+ever, the carbon source for P . ~a~eru9 tn~~osa is glutamate or citrate, valine tnhtbitton of growth does occur : this Tnh~itton is reversed by isoleuctne, pyruvate or by compounds yielding pyruvate, e .g ., glucose or alanine (9) . In P . up tide L-valine also Inhibited growth in glutamate medium, but the effect was transient . Physiological dernpression of leucineand isoleuctne-specific enzymes during the period of transient inhibition implicated isoleuctne-leucine limitation as the basis for inhibition, a result confirmed by the complete reversal of growth Inhibition In the

318

Growth Iahibitioa in Biological Systems

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presence of exogenous leucine and isoleucine . In vitro results with a-IPM synthetase show that L-isoleucine might interfere with L-leucine biosynthesis, while _L-leucine could interfere (threonine deaminase) with L-isoleucine formation . Additionally, false products generated as a result of substrate ambiguities might Inhibit the activity of an enzyme necessary for biosynthesis of leucine, isoIf the basis for inhibition is localized within the leucine or valine . biosynthetic pathway, then inhibition by one end product should be abolished by the presence of the remaining end products . Since the combination of all three amino acids plus pantothenate did not yield the fast growth rate expected, the basis for metabolite inhibition appears to be external to the biosynthetic pathway for branched-chain amino acids . This conclusion is consistent with the finding that none of the mutants selected for resistance to growth inhibition by branched-chain amino acids were regulatory mutants capable of excretion of branched-chain amino acids . Inhibition of unrelated enzymes . Most intermediary metabolites in living cells are present at extremely low intracellular concentrations (unlike , end-product molecules such as amino acids) . Under certain flow-of-carbon patterns, P . p~u~tida may exhibit anomalous Inhibition of unrelated enzymes by branched-chain amino acids or their metabolites . For example, pyruvate metabolism could be disrupted by an accumulated keto acid within the branched-chain amino acid pathway (13), or Coenzyme-A metabolism might be disrupted in some general way owing to effects of accumulated CoA derivatives of branched-chain amino acid degradation . It Is known that in human phenylketonurics, accumulated phenylpyruvate Inhibits glucose-6-phosphate dehydrogenase (16) . In both phenylketonurla and maple syrup urine disease enzymatic capability to oxidize pyruvate and malate is inhibited (13) . Unrelated enzymes inhibited by accumulated metabolites include citrate synthase, fatty acid synthetase, and pyruvate dehydrogenase . Inhibition of glutamate decarboxylase (35) and 2-ketoglutarate dehydrogenase (51) by branched-chain keto acids has also been reported . The entire leucine biosynthetic pathway sequence is exactly analogous to a portion of the TCA cycle . If the TCA cycle enzymes display substrata ambiguities comparable to those of the leucine sequence to P . pu~tida~, it is possible that keto acid Imbalance may limit the efficÏency of TCA cycle function, thereby slowing growth . In point of fact, leucine pathway analogies beginning with a keto acid-acetyl-CoA condensation occur elsewhere, e .g ., a-ketoadipate pathway of lysine biosynthesis . Keto acid imbalances may have the potential to interfere with the efficient function of any of these analogous pathways . Such inhibitory effects are likely to be maximized when the natural substrate of a susceptible enzyme is of particularly low concentration while the inhibitor concentration is abnormally high . The various possibilities considered are not necessarily mutually exclusive . The basis for metabolite inhibition can be extraordinarily complex, and seemingly obvious explanations do not always apply (52) . A tremendous research effort, for example, has impinged upon understanding phenylketonurla (40,53,54) and maple syrup urine disease (13,41) . Nevertheless, the definitive molecular events that explain the clinical syndromes remain controversial .

Vol . 19, No . 3

Growth Inhibition in Biological Systeme References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 . 33 . 34 . 35 . 36 . 37 " 38 .

J .B . STANBURY, J .B . WYNGAARDEN, and D .S . FREDRICKSON, The Metabolic Basis of Inherited Disease , 1778 pp, McGraw-Hill, New Yôrk 1972 . G .W . FRIMPTER, The New Eng . J . Med . 289 895-901 (1973) " J . DOBBING, Pediâtrtcs , 53 2 - 6T974~ J . DOBBING and J .L . SMART, Br . Med . Bull . 30 164-168 (1974) . T .A . TEDESCO, J .W . WU, F .S . BOCHÉS, and W .J.MELLMAN, _The _New End . J . Med . 292 737-740 (1975) " L.OÎNGRAM, . and R .A . JENSEN, Arch . Mtkrobiol . _91 221-233 (1973) " S .C . RITTENBERG, Advanc . Microbtol . Ph stol . _3 159-196 (1969) " G .P . GLADSTONE, J . Ex~ . Path . 20 1 9-(1939) " J .M . VARGA, and Î. HORVATH, J . Bâcteriol . _92 1569 (1966) . H .E . UMBARGER, and B . BROWN, J . Biol . Chem 233 1156-1161 (1958) " 1 . HORVATH, and I . GADO, Acta Microbtol . Acad._Sci . Hun . _12 103to7 (1965) " P . MEURIS, Genetics 63 569-580 (1969) " J .B . CLARK, and J .M . LÂND, Biochem . J . 140 25-29 (1974) " D .H . CALHOUN, and R .A . JENSEN, J . Bacteriôl . 109 365 - 372 (1972 . A .M . CHAKRABARTY, G . CHOU, and Î .C . GUNSALUS, Proc . _Nat . Acad . Sci ., U .S .A . 70 1137-1140 (1973) . .A L . JACOBSON, R .C . BATHOLOMANS, and I .C . GUNSALUS, Biochem . Btophys . Res . Comm . 2 4 955-960 (1966) . O .H . LOWRY,N .J . ROSEBROUGH, A .L . FARR, and R .J . RANDALL, J . Biol . Chem . 192 265-275 (1951) " M. IACCARINO, andP . BERG, J . Bacteriol . 105 527-537 (1970) . R .H . BAUERLE, M . FREUNDLICH, F .C . STORMES, ând H .E . UMBARGER, Blochim . Btophys . Acta 92 142-149 (1964) . W .W . WESTERFIELD, J . Btol. Chem . 161 495-502 (1945) . G .B . KOHLHAW and T.R . LEARY, Methodsin Enzymology , Vol . XVIIA, p . 771, Ed . H . Tabor and C .W . Tabor, Academic Press, Inc ., New York (1970) . E .R . STADTMAN, Methods in Enzymology , Vol . III, Ed . S .P . Colowlck and N .O . Kaplan, Academic Press, New York (1957) " E .J . SIMON and D . SHEMIN, J . Am . Chem . Soc . 75 2520 (1953) " R .T . TAYLOR and W .T . JENKINS, J. Btol . Chem . ~1 4391-5395 (1966) . J . FOLCH, M . LEES and G .H .S . STANLEY, _J . Biol Chem . . _226 497So9 (1957) " L .D . METCALFE and A .A . SCHMITZ, Anal . Chem . 33 363 - 364 (1961) . .H .E . KENNEY, D . .KOMANOWSKY, and A .N . WRIGLEY,J . Amer . OIl Chemists Soc . 42 19-22 (1965) . K . K . CARROLL, Nature 19 377 - 388 (1961) . R .S . CONRAD, L . K . MASSEY, and J .R . SOKATCH, J . Bacteriol . 118 toi-tit (1974) . R .R . MARTIN, V .D . MARSHALL, J .R . SOKATCH, and L . UNGER, J . Bacteriol . 115 198-204 (1973) " K . K . MASSEY, R.S . CONRAD and J .R . SOKATCH, J . Bacteriol . 118 112-120 (1974) . V .D . MARSHALL, and J .R . SOKATCH, J . Bacteriol . 110 1073-1081 (1972) . R . RABIN, I .I . SALAMON, A .S . BLEIWEIS, J . CARLIN, and S .J . AJL, Biochemistry 7 377-387 (1968) " R .M . BORICHEWSKI, J . Bacteriol . 93 597 - 599 (1967) . R .E . TASHIAN, Metabolism 10 3937+02 (1961) . R . SCHREYER and A . BOCK, J.Bacteriol . 115 268-276 (1973) . P .J . WOLFSON and A . KRULWÎCH, J . Bacteriol. 112 356-364 (1972) . E . FREESE, C .W . SHEU and E . GALLIERS, Nature ~1 321-325 (1973) .

320 39 . 40 . 41 . 42 . 43 . 44 . 45 . 46 . 47 . 48 . 49 . 50 . 51 . 52 . 53 . 54 .

Growth Inhibition in Biological Systems

Vol . 19, No . 3

C .W . SHEU, and F . FREESE, J . Baçteriol . 111 516-524 (1972) . G . WEBER, R .I . GLAZER, andR .A . ROSS, Advanc . Enz,! Re ul . _8 13-36 (1969) " M .G . BISSELL, K .G . BENSCH, and M .M . HERMAN, Neurochemistry 22 957-964 (1974) . L .T . HODGINS and V .R . WHEATLEY, Biochlm . Biophys . Acta 316 173179 (1973) . R .L . CONNER and A .E . REILLY, Btochtm . Biophys . Acta 398 209216 (1975) . N .W . CHARON, R .C . JOHNSON, and D . PETERSON, J . Bacteriol . 117 203-211 (1974) . R .E . WEBSTER and S .R . GROSS, Blochemtstr 4 2309-2317 (1965) " D . VOLLBRECHT, Btochtm . Biophys . A~ 382-389 (1974) . F . HILL and H .G . SCHLEGEL, Arch . Mikroblol . 68 1-17 (1969) . J .E . NORTON and J .R . SbKATCH, Bloch~mBiophys . . Acta 206 261269 (1970) . M . FREUNDLICH, and L .P . CLARKS, Blochim . Biophys . Acta 170 271-281 (1968) . R .A . JENSEN and D .L . PIERSON, Nature 254 667-671 (1975) " M .S . PATEL, 8tochem . J . 144 91-97 (197 R .A . JENSEN, S . STENMl1RK-C0X and L .O . INGRAM, J . Bactertol . 120 1124-1132 (1974) . _ F . TAUB and T .C . JOHNSON, Biochem . J . 151 173-180 (1975) " D .M . BRADLEY and R .F . MAHLER, Clin . 8ci Mol Med . 49 -. -. - 343-351 (1975) "