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10 Yeast and Neurospora Invertases
Yeast and Neurospora Invertases J. OLIVER LAMPEN I. Introduction . . . . . . A. Determination . . . . 11. Yeast Invertase . . . . . . A. Localization and Multiple Forms B. Biosynthesis . . . . . C. Preparation of Purified Invertase D. Properties of Purified Eneyme E. Catalytic Properties . . . 111. Neurospora Invertase . . . . A. Purified Preparations . . B. Catalytic Properties . . .
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291
292 292
293 294 295 298
300 303 304 305
1. Introduction
Invertase (EC 3.2.1-26) or P-fructofuranoside fructohydrolase (8-hfructosidase) acts typically on sucrose and related glycosides producing hydrolysis or, in varying degree, fructosyl transfer. The substrate must possess a terminal, unsubstituted P-D-fructofuranosyl residue, but the nature of the “afructon” moiety is of comparatively little importance for the enzymic action. This chapter will deal with the invertases formed by yeast (Saccharomyces species) and by the fungus n’eurospora crassa since these enzymes have been utilized for most recent biochemical and molecular studies. The long history of research on invertase, its function and distribution in nature, its relationship to other sucrose-cleaving enzymes (e.g., inulinases and a-glucosidases) , and many facets of its catalytic action (substrate binding, pH dependence, action of inhibitors, etc.) have 291
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J. OLIVER LAMPEN
all been extensively examined in previous reviews (IS). These features will be discussed only as necessary to the consideration of recent data. Major attention will be devoted to the relatively homogeneous preparations of both yeast and Neurospora enzymes which have become available within the last five years.
A. DETERMINATION Invertase action has been detected by measuring the products of sucrose hydrolysis or of fructosyl transfer. Measurement has usually been carried out either polarimetrically or by determination of the reducing sugar formed ( 4 ) . Recent workers havc often used glucose oxidase to estimate the glucose formed ( 5 , 6) since this procedure is relatively simple, minimizes any error from transfer reactions (which primarily involve the fructosyl moiety), and can be readily automated. II. Yeast Invertase
Yeast invertase has been known the longest of any of the carbohydrases and figured importantly in much of the fundamental work on enzyme kinetics. Thus a great deal of information is available about its catalytic action under a wide variety of conditions and the effects of many different inhibitors. Yet until the last decade no reasonably homogeneous preparations of invertase were available, and very little of the descriptive data could be related to chemical or molecular features of the enzyme. It is no exaggeration to say that the great resistance of the catalytic activity of invertase to destruction during prolonged autolysis has been a major deterrent to elucidation of its molecular nature. Many purified preparations have been obtained from yeast allowed to autolyze for days, and even those subsequently handled very carefully [e.g., Berggren (7) ] are clearly heterogeneous. In addition, 1. K. Myrback, “The Enzymes.” 2nd ed., Vol. 4, p. 374, 1960. 2. C. Neuberg and I. Mandl, “The Enzymes,” 1st ed., Vol. 1, Part 1, p. 527. 1950. 3. A. Gottschalk, “The Enzymes,” 1st ed., Vol. 1, Part 1, p. 577. 1950; Atlumi. Carbohydrate Chem. 5, 49 (1950). 4. S. Hestrin, D. S. Feingold, and M. Schmmm, “Methods in Enzymology,” Vol. 1, p. 231, 1955. 5. S. Gascon and J. 0. Lampen, JBC 243, 1567 (1968). 6. M. Messcr itnd A . Dnhlqvisl, Anal. Biochem. 14, 376 (1906). A. Dalilclvist. A n d . Bwchem. 22, 99 (1968). 7. B. Berggren, Arkiv Kemi 29, 117 (1968).
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AND N E U H O S P O H A I S V E R T A S E S
293
interconversion of two invertase forms has been noted (8) during the extended incubation of homogenates initially prepared rapidly by the use of a French pressure cell. Another potential source of heterogeneity is the existence in yeast of six different genes for invcrtase synthesis [SV,-,, (9, 10)1. The different genes should lead to the production of enzymes which would be similar but not identical. Finally, commercial baker’s and brewer’s yeasts, which are frequently chosen for isolation of invertase since they are readily available in quantity, are heteroploid and contain mixtures of the invertase genes. An autolysate of these yeasts will contain a complex of closely related enzymes which probably have also undergone enzymic alteration (7). It is important that studies on purification and molecular nature of invertase begin with an organism which has only one gene for invertase production (and thus the minimum number of enzyme forms) and that extraction be rapid and conditions selected that will minimize enzymic modification. A. LOCALIZATION AND MULTIPLE FORMS Almost all of the invertase produced by yeast is retained by the intact cell, although a few yeasts do release most of their enzyme (11).Early work, reviewed by Myrback ( I ) , showed that the invertase of intact cells had the same pH-activity curve and sensitivity to inhibitors as enzyme in true solution. The experiments of Preiss ( I d ) , who determined invertase activity of yeast after irradiation with low voltage electrons, indicated that the bulk of the enzyme lies between 500 and 1000 A from the outer edge of the cell, i.e., within the wall itself. More direct proof of an external location for invcrtase (i.e., outside of the permeability barrier of the plasma membrane) was provided by the demonstration that almost all of the activity was released during conversion of the cells to protoplasts with snail gut juice or with microbial enzyme preparations (13-16). A separate form of invertase present only inside the cell membrane 8. J. Hosliino nnd A. Momose, J. Biochem. ( T o k y o ) 59, 192 (1966). 9. 0. Winge and C. Roberts, Compt. Rend. Trav. Lab. Carlaberg, Ser. Phgsiol. 25,
419 (1957). 10. R. K. Mortiiner and D. C. Hawthorne, Genetics 53, 105 (1966). 11. L. J. Wickerham, ABB 76, 439 (1958). 12. J. W. Preiss, ABB 75, 186 (1958). 13. J. Friis and P. Ottolenghi, Compt. Rend. Trav. Lab. Carlsberg 31, 259 (1959). 14. A. A. Eddy and D. H. Williamson, Nature 183, 1101 (1959). 15. D. D. Sutton and J. 0. Lampen, BBA 56, 303 (1962). 16. M. Burger, E. E. Bacon, and J. S. D. Bacon, BJ 78, 504 (1961).
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was detected by Gascon and Ottolenghi (17) using gel filtration on Sephadex G-200 columns. The purified enzyme (6, 18) has a molecular weight of about 135,000 and is free of carbohydrate, in contrast to the larger external enzyme with a molecular weight of 270,000 about half of which is mannan. Beteta and Gascon (19) reported that the small form is present primarily in the yeast vacuole. Small amounts of the large form are also present in the vacuole fraction, but the great bulk of this material is outside the cell membrane. Multiple forms of invertase had previously been demonstrated in cultures of Candida utilis (20) and in yeast autolyzates (8, 21, 2 2 ) ; however, the relation of these materials to the two well-characterized forms is not evident. External invertase can be released from cells of Saccharomyces fragilis and S. rnellis (but not S. cerevisiae) by treatment with thiols (23,2 4 ) , and this has led to the suggestion that the enzyme is retained within a mesh in which disulfide bonds play a critical role (23).Invertase is released from several Saccharomyces strains by phosphomannanase, which removes an outer wall layer of P-diester-linked mannan from the yeast cell, but very little protein or glucan (25, 2 6 ) . Invertase might be bonded to the mannan through similar diester linkages since certain samples of the purified external enzyme contain phosphorous (W. Colonna, personal communication), Retention may also be effected by hydrogen or hydrophilic bonding between the mannan moiety of the invertase and the outer mannan layer, or by direct trapping of the molecule within a mannan mesh. One may conclude that external invertase is held within the wall or between the wall and cell membrane with the structures responsible for its retention varying from one species to another.
B. BIOSYNTHESIS The formation of invertase by yeast and by N . cmssa is pri,marily controlled by catabolite repression, particularly by hexoses. The effects 17. S. Gascon and P. Ottolenghi, Comp. Rend. Trav. Lab. Carlsberg 36, 85 (1967). 18. 5.Gascon, N. P. Neumann, and J. 0. Lampen. JBC 243, 1573 (1968). 19. P. Beteta and S. Gascon, 10th Meeting, Spanish Biochem. SOC.,Madrid. 1970 p. 75. 20. R. Sentandreu, F. Lopez-Belmonte, and J. R. Villanueva, FEBS Absh. p. 28 (1965). 21. E. Cabib, BBA 8, 607 (1952). 22. T.Kaya, J . Agr. Chem. Soc. Japan 38, 417 (1964). 23. D. K. Kidby and R. Davies, BBA 201, 261 (1970). 24. R. Weimberg and W. L. Orton, J . Bacteriol. 91, 1 (1966). 26. W. L. McLellan and J. 0. Lampen, J . Bacteriol. 95, 967 (1968). 26. W. L. McLellan, L. McDaniel. and J. 0. Lampen, J . Bacteriol. 102, 261 (1970).
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of various levels and kinds of sugar have been examined in detail (17, 27-30). A striking feature is that the level of internal invertase in yeast is relatively constant, varying only about threefold with changes in glucose concentration which cause a 1000-fold shift in the activity of the large external enzyme. Thus “high-glucose” yeast contains almost exclusively the internal form (6, 1 7 ) . The biosynthetic relationship between the internal and external invertases has not been clarified. The two enzymes have almost identical kinetic parameters ( K , values, relative V,,, on different substrates and pH-activity curves), low transferase activity, cross-react serologically ; and were both absent in the three sucrose-negative mutants examined (18). The general occurrence of glycosylation during secretion of proteins by yeasts and other fungi (51, 3$) suggests that the internal enzyme may be the precursor of the external glycoprotein, but the reported presence of the internal enzyme in the yeast vacuole [probably the lysosome (SS)]raises the possibility that it is produced by degradation of the glycoprotein form (19). For purification and study of invertase it is convenient to have available an organism which forms the enzyme in high sugar concentrations. A mutant (FH4C) from a Saccharomyces strain (contains only the SU, gene) produces invertase as about 2% of its total protein even when Mutants of N . crussu that are growing in 5% glucose medium (6,S4,36). relatively resistant to catabolite repression have been isolated by Metzenberg ($6) and by Gratsner and Sheehan (37).
C. PREPARATION OF PURIFIED INVERTASE The usual procedures for the extraction and purification of invertase have included a prolonged autolysis to release the enzyme and degrade much of the noninvertase protein, a heat treatment to precipitate the yeast gum (mostly mannan) , and various precipitations or adsorptions R. Davies, BJ 55, 484 (1953). A. Davies, J. Gen.Microbiol. 14, 109 (1956). H. Suomalainen and E. Oura, ABB 88, 425 (1957). F. Dodyk and A. Rothstein, ABB 104, 478 (1984). E. H. Eylar, J . Theoret. B i d . 10, 89 (1965). J. 0. Lampen, Antonie van Leeuwenhoek 3. Mdcrobiol. Serol. 34, 1 (1968). P. Matile and A. Wiemken, Arch. Mikrobiol. 56, 148 (1967). J. 0. Lampen, N. P. Neumann, S. Gascon, and B. S. Montenecourt, in “Organizational Biosynthesis” (H. J. Vogel, J. 0. Lampen, and V. Bryson, eds.), p. 363. Academic Press, New York, 1967. 35. N . P. Neumann and J. 0. Lampen, Biochemistry 6, 468 (1967). 36. R. L. Metsenberg, ABB 98, 468 (1962). 37. H. Gratsner and D. N. Sheehan, J. Baclen’ol. Q7, 544 (1969). 27. 28. 29. 30. 31. 32. 33. 34.
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TABLE I RECENTPREPARATIONS OF YEASTINVERTASE Reference
Carbohy&ate (%I
1951 Fischer and Kohtes
70-80
(98) 1960 Anderson (39) 1965 Myrback and Schilling (40) 1967-1968 Vesterberg and Berggren (7,41) 1967 Neumann and Lampen (36) 1970 Neumann and Lampen (36Y 1968 Gascon and Lampen
I U (30") per mg protein 800-1000
cu.4000
30
3500
1969 Andersen and
Jorgensen (43) 1969 Greiling et al. (49) 1971 Waheed and Shall (44)
A
A
112,000
A or M
270 ,000 270,000
A
30-40
ca. 2700-3000
50
2700-3001)s
M
47-52
4000-5OW
M
<3
1970 Gascon and Lampen
(0
Method of Molecular extraction4 weight
2901)s
M
cu.3501)s
M
13
3780
A
77 50
1600 2771)s
A A
135,000
A, autolysis; M, mechanical breakage.
* Homogeneous by gel electrophoresis.
Unpublished results; author's laboratory. Internal enzyme; others primarily external.
of the enzyme ( 1 , 2 ) . More recent attempts have utilized gel filtration, ion exchange chromatography, gel electrophoresis, and electrofocusing. Some of these preparations are listed in Table I (5, 7, 35, 38-44) with the reported specific activities converted to international units (IU) a t 30" per mg of protein. The assignments of activity a t 30" are somewhat arbitrary since the temperature coefficients reported vary in several instances. The best fractions tend to have a specific activity of about 38. E. H. Fischer and L. Kohtes, Helv. Chim. Aclu 34, 1123 (1951). 39. B. Andersen. Acta Chem. Scand. 14, 1849 (1960). 40. K. Myrback nnd W. Schilling, Enzymologin 29, 306 (1965). 41. 0. Vesterberg and B. Berggren, Arkiv Kemi 27, 119 (1967). 42. B. Andenen and 0. 5. Jorgensen, Acta Chem. Scund. 23, 2270 (1969). 43. H. Greiling, P. Vogele, R. Esters, and H.-D. Ohlenbusch, Z. Physiol. Chem. 350, 517 (1969). 44. A . Waheed and S. Shall, FEBS Abslr. No. 481 (1967); BBA 242, 172 (1971).
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YEAST AND NEUROSPORA INVERTASES
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4000 IU/mg protein, although individual preparations have exceeded 5000 IU. It should be noted that Neumann and Lampen (56)used heating and ethanol precipitation and regularly obtained apparently homogeneous material with a specific activity of about 3000 IU. After elimination of these steps, potencies of 4000 to 5000 IU have been routine. This suggests that a significant amount of inactivation occurs during these treatments and that the partially inactive material subsequently fractionates with the native enzyme. Many of the purified preparations (especially those in which the invertase was extracted rapidly rather than by prolonged autolysis) contain about 50% carbohydrate. Neumann and Lampen (36,&) found that the mannan is covalently attached to the protein moiety of the enzyme, probably through glucosaminyl-asparagine bonds. The carbohydrate of Berggren’s (7) purified invertase was not removed by dimeriaation with Hgz+ or by gel filtration in 4 M urea and 0.1 M mercaptoethanol ; the mannan and protein were considered to be covalently linked. Greiling et aZ. (43) reported that their invertase preparation contained mannan linked to serine or threonine by O-glycosidic bonds. Few, if any, such alkali-labile linkages were detected by Neumann and Lampen (46) although this type of linkage is common in the bulk structural mannan of the yeast wall (46). Since the enzyme studied by Greiling et aZ. (49) contained almost 80% mannan and no evidence of homogeneity was presented, the 0-glycosides may have been present in mannan peptides not linked to the invertase molecules. Invertase samples purified by procedures which include prolonged autolysis frequently contain low amounts of carbohydrate, especially if the enzyme has been collected by precipitation with ammonium sulfate (42, 47). [The carbohydrate-free internal enzyme is readily precipitated in 70% saturated ammonium sulfate (6) ; the external glycoprotein enzyme is relatively soluble even in saturated ammonium sulfate (36).] In one instance (48) the mannan could be removed from the preparation by treatment with bentonite a t pH 2.9 leaving an unstable enzyme; the carbohydrate-free internal invertase is also unstable a t this pH (18). The best working hypothesis is that external invertase is a glycoprotein containing about 50% mannan and 2 4 % glucosamine (probably the N-acetyl form). During prolonged autolysis much of the mannan can be removed, leaving a heterogeneous enzyme with a low or even negligible mannan content. 45. N. P. Neumann and J. 0. Lampen, Biochembtly 8, 3552 (1969). 46. R. Sentandreu and D. H. Northcote, BJ 109, 419 (1968). 47, M. Adams and C. S. Hudson, JAC8 65, 1359 (1943). 48. E. H. Fischer, L. Kohtes, and J. Fellig, Helv. Chim. Acta 34, 1132 (1951)
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The carbohydrate moiety of external invertase is not essential for its catalytic activity since several highly active preparations have been almost devoid of carbohydrate (42,47, 48). Also, the internal invertase has a t least two-thirds the specific activity of the best external preparations [the value of this comparison is limited, however, since the amino acid compositions of the internal and external enzymes differ strikingly in the single strain examined (18)1. The carbohydrate moiety does increase thc stability of the external enzyme to a variety of agents. Arnold (49) has shown that susceptibility to heat inactivation decreases with increasing carbohydrate level. The internal (carbohydrate-free) enzyme is more sensitive to acid pH than is the external enzyme (18), although it is more stable a t alkaline pH. Also, the external enzyme is unaffected by condensed tannins, whereas the internal enzyme is sensitive (60).
D. PROPERTIES OF PURIFIED ENZYME Most of the external invertase preparations of high specific activity listed in Table I were reasonably homogeneous in that they gave a single band upon polyacrylamide gel electrophoresis and have usually shown single though broad peaks upon gel filtration or ion exchange chromatography. The enzyme from Saccharomyces mutant FH4C (56) is free of substantial contamination with nucleic acid (A2so:A260 = 1.8) and has an Eig (based on protein content) of 23.0. On ultracentrifugaof 10.4s (molecular weight tion it showed a major component with s20,w 270,000 i= 11,000 daltons) and a minor peak, probably a dimer, which mas also enzymically active. Material from the main peak did not aggregate when rerun under the same conditions; in contrast, the purified invertase from Saccharomyces strain LK2G12 aggregates readily and reversibly (S5). Although the specific activity per mg protein is reasonably uniform across tlic major pcak from ion excliangc chromatography (35),the carbohydrate content usually shifts, e.g., from 52 to 53% a t the front of the peak to 47-48% at the back. This variation may result from partial enzymic removal of the mannan even during rapid purification, or the size of the carbohydratc side chains may vary naturally. I n the one preparation examined (45) approximately 30 chains of mannan with a size range of 2,000-10,000 daltons were present per molecule of enzyme, linked to the protein through glucosaminyl-asparagine bonds. The 49. W. N. h n o l d . BBA 178, 247 (1969). 50. D. H. Strumeyer and M. J. Malin, BJ 118, 899 (1970).
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299
AND NEUROSPORA IKVERTASES
mannan associated with invertase has been reported (61) to be identical to the bulk mannan of the yeast wall, but the mannan of FH4C invertase (46) differs a t least in that it contains few, if any, of the serine- or threonine-0-mannose linkages characteristic of the wall mannan (43, 46). The amino acid compositions of external invertase from brewer's yeast (42) and mutant FH4C (18) and of the internal enzyme from the mutant (18) are compared in Table 11. The distribution of amino acids in the two external enzymes is similar and contrasts sharply with the internal enzyme, especially in the levels of glycine, tyrosine, histidine, and halfcystine. The external enzyme from mutant FH4C was not activated by cysteine (18) [although Hoshino and Momose (62)did report a fourfold TABLE I1 AMINOACIDCOMPOSITION OF PURIFIED INTERNAL AND EXTEBNAL INVERTABES Molee/135,000 g of protein
External Amino acid
[Ref. (IS)]
Glycine Alanine Serine Threonine Proline Valiie Isoleucine Leucine Phenylalanine Tyrosine Tryptophan Half-cystine Methionine Aspartic acid Glutamic acid Arginine Histidine Lysine Glucosamine
71 68 114 84 65 69 40 83 80 65 33 5 21 178 115 27
(Mannose)
(50%)
a
16
60 38
Internal [Ref. (U)] 74 72 129 106
115 84
a
151 80 63 73 38 77 77 31 30 0 14 165 124 32 29 85 0
(13%)
(<3%)
64 68
39 76
85 71 a
>1 19 171 116 27 16 54
Not determined.
51. A. J. Cifonelli and F. Smith, JACS 7'7, 5682 (1955). 52. J. Hoshino and A. Momose, J . Gen. A p p l . Microbid. 12, 163 (1966).
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activation of two forms from baker’s yeast], nor was it sensitive to or reactive with sulfhydryl reagents (36).Three SH groups were accessible to iodoacetate in 8 M urea (35) ; the remaining two residues may exist as a disulfide bridge. The pH-stability curves for the external and internal forms differ markedly (18). The external enzyme is inactivated a t pH 8 and above, but it is stable at pH 3. The internal form is stable a t pH 9, but it undergoes reversible inactivation at pH 5 or less. During this inactivation there was no change in the molecular weight of the internal enzyme.
E. CATALYTIC PROPERTIW 1. Active Site
To be cleaved by yeast invertase a substrate must contain an unsubstituted P-D-fructofuranosyl residue (1, 3 ) . Gottschalk (3) has concluded that the enzyme combines primarily with this moiety through the glycosidic oxygen and the OH groups a t C-6 and C-3 of fructose. There may be some specific interaction with the OH a t C-2 of glucose since sucrose is the best substrate. Large “afructon” groups decrease the rate of cleavage, possibly through steric factors. Rupture of the glycosidic linkage occurs on the fructose side of the glycosidic oxygen (53, 6 4 ) . Binding of the substrate involves a group(s) with pK c 3 (55-57) which determines the acid side of the pH-activity curve. The most attractive possibility is a carboxylic group. The alkaline side of the pH-activity curve has the form of a dissociation curve of a weak acid with pK z 7, probably the imidazole group of a histidine residue ( I , 5 8 ) . This residue does not appear to be involved in substrate binding since the alkaline side of the pH curve is not dependent on substrate concentration (59) [such a dependence has clearly been demonstrated for the acid limb ( G O ) ] . Thus the active form of the enzyme requires at least an unfor substrate binding and a proprotonated acidic group (COO-?) tonated imidazole residue which is apparently involved in substrate cleavage. 53. D.E.Koshlnnd, Jr., and S. S. Stein, JBC 208, 139 (1954). 54. D.E. Koshland, Jr., Biol. Rev. 28, 416 (1953). 55. K. Myrback, 2. Physiol. Chem. 158, 160 (1926). 56. K. Myrback, SOC. Biol. Chemists, India, Silver Jubilee Souvenir p. 204 (1956). 57. K.Myrback and E. Willstaedt, Arkiv Kemi 15, 379 (1960). 58. S. Shall and A. Waheed. BJ 111, 33P (1969). 59. R. Kuhn, 2.Physiol. Chem. 125, 28 (1923). 60. K. Myrback and U. Bjorklund, Arkiv Kemi 4, 567 (1952).
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YEAST AND NEC'ROSPORA ISTERTASES
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Sulfhydryl Groups. It has frequently been suggested that an SH group participates in the catalytic action of invertase. The activity of some preparations can be increased several fold by cysteine (529, and the enzyme can be inactivated by iodine and by Hg2+ or Ag2+ ( l ) ,although not by the more specific SH reagents (35). Also the iodine-invertase produced at pH 5.0 (which retains about 50% of the original activity) is much more resistant to Hg" o U g ' + tha9 is the native enzyme. This led to the suggestion that iodine reacts at pH 5.0 with an SH group that is partially masked in the native form of invertase (1). The concept has been questioned since cysteine, glutathione, awl ascorbic acid do not produce reactivation (61), but it was supported by the observation of Shall and Waheed (58) that nonacidic thiols (mercaptoethanol and mercaptoethylamine) were effective. The latter workers (58) proposed that lack of reactivation by cysteine resulted from charge repulsion by a COOgroup a t or near the active center. , The mechanism of iodine inactivation and indeed the question of any catalytic role for the SH groups of external invertase must, however, be left undecided in light of the fwt that internal invertase lacks cysteine residues yet shows catalytic properties) very similar to those of the external enzyme (18). (Recent studies by Q.Waheed in the author's laboratory have shown that internal invertase also reacts with iodine to yield a product with approximately half the original activity.) The most tenable assumption for the present is that iodine does not react with an SH of the external enzyme [nor with , a tyrosine or histidine residue (58)] but possibly oxidizes a methionine residue to the sulfoxide [cf. Koshland et al. (62, 6S)I. 2. Inhibitors
Inhibition of invertase by Znz+is reversible and noncompetitive, and it is strongly dependent on p H ( 6 4 ) . The reactive group on the protein appears to be the unprotonated form of the imidazole group determining the alkaline side of the pH-activity curve. Myrback (64) proposed that Zn2+ (and Hg2+)links two enzyme molebules to form a dimer. Dimerization by Hgz+ has been demonstrated by Berggren (Y), who attempted unsuccessfully to separate the enzyme protein and the mannan in this manner. 61. K. Myrback and E. Willstaedt, Arkiv Kemi 13, 179 (1957). 62. D. E. Koshland, Jr., D. H. Strumeyer, and W. J. Ray, Jr., Brookhaven Sump. Biol. 15, 101 (1962). 63. M . E. Koshland, F. M. Englberger, M. J. Erwin, and S. M. Gaddone, JBC 238, 1343 (1963). 64. K. Myrback, Arkiv Kemi 27, 507 (1967).
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J . OLIVER LAMPEN
Pyridoxal and pyridoxal phosphate are potent inhibitors of potato invertase (66), but they are much less effective against the yeast or Neurospora enzymes. I n contrast, the fungal enzymes are very sensitive to inhibition by aniline whereas the potato enzyme is resistant (65, 6 6 ) . Invertase undergoes i,mmediate reversible inactivation by concentrations of urea which do not produce major changes in physical properties (67). It appears to combine with ca. 1.3 molecules of urea per catalytic site (68). At high urea concentrations, e.g., 8 M at p H 5.0, or a t pH values above 6.0, irreversible inactivation occurs along with changes in secondary and tertiary structure. 3. Kinetics
The values of K , for soluble invertase and for the enzyme bound to cell walls were identical [determined from plots of the inhibition by aniline ( 6 9 ) ] .The K , for the wall preparations was lower (33 mM) than for the soluble enzyme (44 mM) ; this was considered to reflect some hindrance imposed by the site of the bound enzyme. Invertase insolubilized by ionic binding to DEAE-cellulose was prepared by Suzuki e t nl. (70). The apparent pH optimum was displaced from pH 5.4 for the free enzyme to pH 3.4 for the bound; however, the latter value is probably not a true optimum pH since the DEAE-cellulose would probably provide a microenvironment of higher pH than that of the external buffer. 4. Mechanism
Following discovery of the activity of yeast invertase in transferring P-fructofuranosyl units to primary alcohols [for references, cf. Myrbiick ( I ) ] , it has frequently been proposed that a two-step reaction occurs with an active enzyme-fructose complex as the first step. This can be written as IEH+GOFsEF+GOH I1 EF HOH s EH FOH I11 EF XOH e EH XOF
+ +
+ +
65. R. Pressey, BBA 159, 414 (1968). 66. K. Myrbiick, 2. Physiol. Chem. 158, 160 (1926). 67. A. M. Chase and M. S. Krotkov, J . Cellular Comp. Physiol. 47, 305 (1956). 68. A. M. Chase, H. C. von Meier, and V. J. Menna, J . Cellular Comp. Physiol. 59, 1 (1962). 69. M. V. Tracey, BBA 77, 147 (1963). 70. H. Sueuki, Y. Oeawa, H. Maeda, and 0. Tanabe, R e p t . Ferment. Res. Inst. 31, 11 (1967).
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YEAST AND NEUROSPORA INVERTASES
303
where E stands for enzyme, GOF for sucrose, GOH and FOH for glucose and fructose, and XOH any receptor sugar. Andersen (71) has recently shown that high concentrations of free fructose (but not glucose) can act as both donor and acceptor giving rise to fructose disaccharides. This provides strong support for the existence of an active enzyme-fructose complex. The qualitative and quantitative distribution of mono-, di-, tri-, and tetrasaccharides during sucrose cleavage is also consistent with a twostep reaction mechanism in which the first step is the formation of an enzyme-fructose complex (72). 111. Neurospora Invertare
The invertase of N . crussa has many features in common with that from Saccharomyces yeasts. Most of the enzyme is retained by the fungal cell and is external to the cell membrane; thus, it is freely accessible to exogenous substrates and has kinetic parameters and sensitivity t o inhibitors identical with those of the free enzyme (73). By a variety of biochemical and histochemical proceduies, the cell-bound enzyme has been shown to be partly in the cell wall and partly between the wall and the cell membrane (73-76). A portion of the enzyme is bound very tightly by the wall and can be extracted only with difficulty (73, 75). Two sizes of active enzyme are present in Neurospora (77, 78), and there is a preferential release of the smaller form during growth (79). Since Manocha and Colvin (80) reported the occurrence of discrete pores in the cell wall of Neurospora, it has been suggested that release of the external enzyme occurs through these pores with molecular sieving favoring the passage of the smaller form. The two sizes of Neurospora invertase are not differentially distributed as are those of yeast. Heavy (H) enzyme predominates both inside Neurospora protoplasts and in the walls of growing cells, but the light 71. B. Andersen, Acta Chem. Scand. 21, 828'(1967). 72. B. Andersen, N. Thiesen, and P. E. Broe, Acta Chem. Scand. 23, 2367 (1969). 73. R. L. Metsenberg, BBA 74, 455 (1963). 74. E. P. Hill and A. S. Sussman, J . Bacteriol. 88, 1556 (1964). 75. M. L. Sargent and D. 0. Woodward, J . Bacteriol. 97, 867 (1969). 76. P. L. Y. Chung and J. R. Trevithick, J . Bacte+l. 102, 423 (1970). 77. F. I. Eilers, J. Allen, E. P. Hill, and A. S. Sussman, J . Hislochem. Cytochem. 12, 448 (1964). 78. R. L. Metsenberg, ABB 100, 503 (1963). 79. J. R. Trevithick and R. L. Metsenberg, J . Bacteriol. 92, 1010 and 1016 (1968). 80. M. Manocha and J. Colvin, J . Bacten'ol. 94, 202 (1967).
304
J. OLIVEB LAMPEN
(L) enzyme is always present in small amounts (81, 82). The L enzyme is favored at low pH and can be produced from the H form by heating or by high salt concentration either a t acid or alkaline pH (82). It reaggregates in concentrated solutions in the absence of salt (e.g., during dialysis of an ammonium sulfate precipitate). A. PURIFIED PREPARATIONS Neurospora crassa invertase was first obtained in a homogeneous state by Metzenberg (78) who purified the material extractable from mycelial powder by pH 5.0 buffer. The preparation was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. It contained 2.4% hexosamine but no detectable neutral sugar. Shortly thereafter Eilers et al. (77) noted that crude extracts of N . crassa gave two bands of activity by gel electrophoresis. This observation was confirmed by Metzenberg (82) and the two components separated by gel filtration into heavy (H) and light (L) forms. Typically 65435% of the activity was in the H form. The szo,wvalues for the H and L enzymes were 10.3 and 5.2, respectively. As already noted the H form can be converted to L enzyme by heat or high salt. During dissociation a transient form with s20,w= 9.8 could be detected. This was considered to be the polymeric enzyme in a preclastic conformation (82). From Metzenberg’s data it can be suggested that Neurospora invertase occurs primarily as the polymeric H form which dissociates under appropriate conditions to yield four active subunits (if both molecules are assumed to be spherical). No evidence was obtained for more than one type of subunit (82). The L enzyme obtained by dissociation of the H enzyme seems to be identical to that present in cell extracts. Dissociation in vitro caused 31% loss of activity; but there was no change of activity during reaggregation, so that the true activity of the subunits may be the same in monomeric and in polymeric form. Solutions of L invertasc foamed copiously upon shaking, whereas H invertase showed little tendency to foam (82). This was considered to indicate that the hydrophobic side chains of H invertase are oriented inwardly and become exposed during dissociation. According to this concept hydrophobic bondings might serve to maintain the polymeric structure of the H form. Pure invertase has recently been isolated from another high-producing strain of N . crassa (37) by Meachum et al. (83).The striking charac81. J. R. Trevithick and R . L. Metzenberg, BBRC 16, 319 (1964). 82. R. L. Metzenberg, BBA 89, 291 (1964). 83. Z. D. Meachum, Jr., H. J. Colvin, Jr., and H. D. Braymer, Biochemistry 10, 326 (1971).
10. YEAST
AND NEUROSPORA INVERTASES
305
teristic of this material is that it contains approximately 11% mannose and 3% glucosamine covalently linked to the protein moiety. Proteinase digestion yielded peptides containing glucosamine and aspartic acid, suggesting that the glucosaminyl-asparagine bond predominates here as in yeast external invertase (45).If serine-0-glycoside linkages are present, their number must be small. The isolated enzyme had an s,O,,, of 10.5 and a molecular weight of 210,000. Four disulfide bonds were detected per mole. In the presence of a dissociating agent (6 M guanidine) and under reducing conditions, the s20,wwas 5.2 and the molecular weight 51,500.The native enzyme thus appears to be a tetramer and, from tryptic peptide fingerprints, probably contains more than one type of subunit. As with Metzenberg's strain (82) both the 10.5 S and 5.2 S forms are found in crude cell extracts and both are enzymically active. The specific activity of Braymer's (83) purified enzyme is 1820 pmoles of sucrose split per minute per milligram of glycoprotein; the value for Metzenberg's material (78) is 1890 poles/minute.
B . CATALYTIC PROPERTIES The substrate specificity of Neurospora invertase (78) is similar to that of the yeast enzyme. Sucrose is the most readily utilized substrate with a K,, of 6.1 x 10-3M and the highest VMx. For raffinose, K,,, is 6.5 x W S M and the V , , , 20% of that for sucrose. The values for &methyl fructoside are K , of 3.3 x lo-* M and relative V,,, of 30%. [These K , values are notably lower than the 26 X M for sucrose and 150 x M for raffinose obtained with the purified yeast enzyme (18).] Trehalose and melezitose were not attacked. The activation energies (calculated from rates a t 0" and 38") are 10.8, 12.3, and 13.9 kcal for sucrose, raffinose, and P-methyl fructoside, respectively. The pH-activity curve has a broad optimum at pH 4.5-6.0. The alkaline limb approximates that for yeast invertase, but the acid side falls off rapidly below pH 4.5 (78). Neurospora invertase is less sensitive than the yeast enzyme to inhibition by divalent cations. Zn2+and Cu2+caused only 33 and 79% inhibition, respectively, a t 0.05M . Both enzymes are strongly inhibited by aniline (65, 78). The sulfhydryl reagent p-hydroxymercuribenzoate inhibits the fungal enzyme and partial protection is afforded by the presence of substrate (sucrose). In contrast the purified yeast external enzyme was not inhibited by several sulfhydryl reagents, including p-mercuribenzoate (35).
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
. . . . . . .
. . . . . . .
291
292 292
293 294 295 298
300 303 304 305
1. Introduction
Invertase (EC 3.2.1-26) or P-fructofuranoside fructohydrolase (8-hfructosidase) acts typically on sucrose and related glycosides producing hydrolysis or, in varying degree, fructosyl transfer. The substrate must possess a terminal, unsubstituted P-D-fructofuranosyl residue, but the nature of the “afructon” moiety is of comparatively little importance for the enzymic action. This chapter will deal with the invertases formed by yeast (Saccharomyces species) and by the fungus n’eurospora crassa since these enzymes have been utilized for most recent biochemical and molecular studies. The long history of research on invertase, its function and distribution in nature, its relationship to other sucrose-cleaving enzymes (e.g., inulinases and a-glucosidases) , and many facets of its catalytic action (substrate binding, pH dependence, action of inhibitors, etc.) have 291
292
J. OLIVER LAMPEN
all been extensively examined in previous reviews (IS). These features will be discussed only as necessary to the consideration of recent data. Major attention will be devoted to the relatively homogeneous preparations of both yeast and Neurospora enzymes which have become available within the last five years.
A. DETERMINATION Invertase action has been detected by measuring the products of sucrose hydrolysis or of fructosyl transfer. Measurement has usually been carried out either polarimetrically or by determination of the reducing sugar formed ( 4 ) . Recent workers havc often used glucose oxidase to estimate the glucose formed ( 5 , 6) since this procedure is relatively simple, minimizes any error from transfer reactions (which primarily involve the fructosyl moiety), and can be readily automated. II. Yeast Invertase
Yeast invertase has been known the longest of any of the carbohydrases and figured importantly in much of the fundamental work on enzyme kinetics. Thus a great deal of information is available about its catalytic action under a wide variety of conditions and the effects of many different inhibitors. Yet until the last decade no reasonably homogeneous preparations of invertase were available, and very little of the descriptive data could be related to chemical or molecular features of the enzyme. It is no exaggeration to say that the great resistance of the catalytic activity of invertase to destruction during prolonged autolysis has been a major deterrent to elucidation of its molecular nature. Many purified preparations have been obtained from yeast allowed to autolyze for days, and even those subsequently handled very carefully [e.g., Berggren (7) ] are clearly heterogeneous. In addition, 1. K. Myrback, “The Enzymes.” 2nd ed., Vol. 4, p. 374, 1960. 2. C. Neuberg and I. Mandl, “The Enzymes,” 1st ed., Vol. 1, Part 1, p. 527. 1950. 3. A. Gottschalk, “The Enzymes,” 1st ed., Vol. 1, Part 1, p. 577. 1950; Atlumi. Carbohydrate Chem. 5, 49 (1950). 4. S. Hestrin, D. S. Feingold, and M. Schmmm, “Methods in Enzymology,” Vol. 1, p. 231, 1955. 5. S. Gascon and J. 0. Lampen, JBC 243, 1567 (1968). 6. M. Messcr itnd A . Dnhlqvisl, Anal. Biochem. 14, 376 (1906). A. Dalilclvist. A n d . Bwchem. 22, 99 (1968). 7. B. Berggren, Arkiv Kemi 29, 117 (1968).
10. Y EA S T
AND N E U H O S P O H A I S V E R T A S E S
293
interconversion of two invertase forms has been noted (8) during the extended incubation of homogenates initially prepared rapidly by the use of a French pressure cell. Another potential source of heterogeneity is the existence in yeast of six different genes for invcrtase synthesis [SV,-,, (9, 10)1. The different genes should lead to the production of enzymes which would be similar but not identical. Finally, commercial baker’s and brewer’s yeasts, which are frequently chosen for isolation of invertase since they are readily available in quantity, are heteroploid and contain mixtures of the invertase genes. An autolysate of these yeasts will contain a complex of closely related enzymes which probably have also undergone enzymic alteration (7). It is important that studies on purification and molecular nature of invertase begin with an organism which has only one gene for invertase production (and thus the minimum number of enzyme forms) and that extraction be rapid and conditions selected that will minimize enzymic modification. A. LOCALIZATION AND MULTIPLE FORMS Almost all of the invertase produced by yeast is retained by the intact cell, although a few yeasts do release most of their enzyme (11).Early work, reviewed by Myrback ( I ) , showed that the invertase of intact cells had the same pH-activity curve and sensitivity to inhibitors as enzyme in true solution. The experiments of Preiss ( I d ) , who determined invertase activity of yeast after irradiation with low voltage electrons, indicated that the bulk of the enzyme lies between 500 and 1000 A from the outer edge of the cell, i.e., within the wall itself. More direct proof of an external location for invcrtase (i.e., outside of the permeability barrier of the plasma membrane) was provided by the demonstration that almost all of the activity was released during conversion of the cells to protoplasts with snail gut juice or with microbial enzyme preparations (13-16). A separate form of invertase present only inside the cell membrane 8. J. Hosliino nnd A. Momose, J. Biochem. ( T o k y o ) 59, 192 (1966). 9. 0. Winge and C. Roberts, Compt. Rend. Trav. Lab. Carlaberg, Ser. Phgsiol. 25,
419 (1957). 10. R. K. Mortiiner and D. C. Hawthorne, Genetics 53, 105 (1966). 11. L. J. Wickerham, ABB 76, 439 (1958). 12. J. W. Preiss, ABB 75, 186 (1958). 13. J. Friis and P. Ottolenghi, Compt. Rend. Trav. Lab. Carlsberg 31, 259 (1959). 14. A. A. Eddy and D. H. Williamson, Nature 183, 1101 (1959). 15. D. D. Sutton and J. 0. Lampen, BBA 56, 303 (1962). 16. M. Burger, E. E. Bacon, and J. S. D. Bacon, BJ 78, 504 (1961).
294
J . OLIVER LAMPEN
was detected by Gascon and Ottolenghi (17) using gel filtration on Sephadex G-200 columns. The purified enzyme (6, 18) has a molecular weight of about 135,000 and is free of carbohydrate, in contrast to the larger external enzyme with a molecular weight of 270,000 about half of which is mannan. Beteta and Gascon (19) reported that the small form is present primarily in the yeast vacuole. Small amounts of the large form are also present in the vacuole fraction, but the great bulk of this material is outside the cell membrane. Multiple forms of invertase had previously been demonstrated in cultures of Candida utilis (20) and in yeast autolyzates (8, 21, 2 2 ) ; however, the relation of these materials to the two well-characterized forms is not evident. External invertase can be released from cells of Saccharomyces fragilis and S. rnellis (but not S. cerevisiae) by treatment with thiols (23,2 4 ) , and this has led to the suggestion that the enzyme is retained within a mesh in which disulfide bonds play a critical role (23).Invertase is released from several Saccharomyces strains by phosphomannanase, which removes an outer wall layer of P-diester-linked mannan from the yeast cell, but very little protein or glucan (25, 2 6 ) . Invertase might be bonded to the mannan through similar diester linkages since certain samples of the purified external enzyme contain phosphorous (W. Colonna, personal communication), Retention may also be effected by hydrogen or hydrophilic bonding between the mannan moiety of the invertase and the outer mannan layer, or by direct trapping of the molecule within a mannan mesh. One may conclude that external invertase is held within the wall or between the wall and cell membrane with the structures responsible for its retention varying from one species to another.
B. BIOSYNTHESIS The formation of invertase by yeast and by N . cmssa is pri,marily controlled by catabolite repression, particularly by hexoses. The effects 17. S. Gascon and P. Ottolenghi, Comp. Rend. Trav. Lab. Carlsberg 36, 85 (1967). 18. 5.Gascon, N. P. Neumann, and J. 0. Lampen. JBC 243, 1573 (1968). 19. P. Beteta and S. Gascon, 10th Meeting, Spanish Biochem. SOC.,Madrid. 1970 p. 75. 20. R. Sentandreu, F. Lopez-Belmonte, and J. R. Villanueva, FEBS Absh. p. 28 (1965). 21. E. Cabib, BBA 8, 607 (1952). 22. T.Kaya, J . Agr. Chem. Soc. Japan 38, 417 (1964). 23. D. K. Kidby and R. Davies, BBA 201, 261 (1970). 24. R. Weimberg and W. L. Orton, J . Bacteriol. 91, 1 (1966). 26. W. L. McLellan and J. 0. Lampen, J . Bacteriol. 95, 967 (1968). 26. W. L. McLellan, L. McDaniel. and J. 0. Lampen, J . Bacteriol. 102, 261 (1970).
10.
YEAST AND NEUROSPORA INVERTASES
295
of various levels and kinds of sugar have been examined in detail (17, 27-30). A striking feature is that the level of internal invertase in yeast is relatively constant, varying only about threefold with changes in glucose concentration which cause a 1000-fold shift in the activity of the large external enzyme. Thus “high-glucose” yeast contains almost exclusively the internal form (6, 1 7 ) . The biosynthetic relationship between the internal and external invertases has not been clarified. The two enzymes have almost identical kinetic parameters ( K , values, relative V,,, on different substrates and pH-activity curves), low transferase activity, cross-react serologically ; and were both absent in the three sucrose-negative mutants examined (18). The general occurrence of glycosylation during secretion of proteins by yeasts and other fungi (51, 3$) suggests that the internal enzyme may be the precursor of the external glycoprotein, but the reported presence of the internal enzyme in the yeast vacuole [probably the lysosome (SS)]raises the possibility that it is produced by degradation of the glycoprotein form (19). For purification and study of invertase it is convenient to have available an organism which forms the enzyme in high sugar concentrations. A mutant (FH4C) from a Saccharomyces strain (contains only the SU, gene) produces invertase as about 2% of its total protein even when Mutants of N . crussu that are growing in 5% glucose medium (6,S4,36). relatively resistant to catabolite repression have been isolated by Metzenberg ($6) and by Gratsner and Sheehan (37).
C. PREPARATION OF PURIFIED INVERTASE The usual procedures for the extraction and purification of invertase have included a prolonged autolysis to release the enzyme and degrade much of the noninvertase protein, a heat treatment to precipitate the yeast gum (mostly mannan) , and various precipitations or adsorptions R. Davies, BJ 55, 484 (1953). A. Davies, J. Gen.Microbiol. 14, 109 (1956). H. Suomalainen and E. Oura, ABB 88, 425 (1957). F. Dodyk and A. Rothstein, ABB 104, 478 (1984). E. H. Eylar, J . Theoret. B i d . 10, 89 (1965). J. 0. Lampen, Antonie van Leeuwenhoek 3. Mdcrobiol. Serol. 34, 1 (1968). P. Matile and A. Wiemken, Arch. Mikrobiol. 56, 148 (1967). J. 0. Lampen, N. P. Neumann, S. Gascon, and B. S. Montenecourt, in “Organizational Biosynthesis” (H. J. Vogel, J. 0. Lampen, and V. Bryson, eds.), p. 363. Academic Press, New York, 1967. 35. N . P. Neumann and J. 0. Lampen, Biochemistry 6, 468 (1967). 36. R. L. Metsenberg, ABB 98, 468 (1962). 37. H. Gratsner and D. N. Sheehan, J. Baclen’ol. Q7, 544 (1969). 27. 28. 29. 30. 31. 32. 33. 34.
296
J. OLIVER LAMPEN
TABLE I RECENTPREPARATIONS OF YEASTINVERTASE Reference
Carbohy&ate (%I
1951 Fischer and Kohtes
70-80
(98) 1960 Anderson (39) 1965 Myrback and Schilling (40) 1967-1968 Vesterberg and Berggren (7,41) 1967 Neumann and Lampen (36) 1970 Neumann and Lampen (36Y 1968 Gascon and Lampen
I U (30") per mg protein 800-1000
cu.4000
30
3500
1969 Andersen and
Jorgensen (43) 1969 Greiling et al. (49) 1971 Waheed and Shall (44)
A
A
112,000
A or M
270 ,000 270,000
A
30-40
ca. 2700-3000
50
2700-3001)s
M
47-52
4000-5OW
M
<3
1970 Gascon and Lampen
(0
Method of Molecular extraction4 weight
2901)s
M
cu.3501)s
M
13
3780
A
77 50
1600 2771)s
A A
135,000
A, autolysis; M, mechanical breakage.
* Homogeneous by gel electrophoresis.
Unpublished results; author's laboratory. Internal enzyme; others primarily external.
of the enzyme ( 1 , 2 ) . More recent attempts have utilized gel filtration, ion exchange chromatography, gel electrophoresis, and electrofocusing. Some of these preparations are listed in Table I (5, 7, 35, 38-44) with the reported specific activities converted to international units (IU) a t 30" per mg of protein. The assignments of activity a t 30" are somewhat arbitrary since the temperature coefficients reported vary in several instances. The best fractions tend to have a specific activity of about 38. E. H. Fischer and L. Kohtes, Helv. Chim. Aclu 34, 1123 (1951). 39. B. Andersen. Acta Chem. Scand. 14, 1849 (1960). 40. K. Myrback nnd W. Schilling, Enzymologin 29, 306 (1965). 41. 0. Vesterberg and B. Berggren, Arkiv Kemi 27, 119 (1967). 42. B. Andenen and 0. 5. Jorgensen, Acta Chem. Scund. 23, 2270 (1969). 43. H. Greiling, P. Vogele, R. Esters, and H.-D. Ohlenbusch, Z. Physiol. Chem. 350, 517 (1969). 44. A . Waheed and S. Shall, FEBS Abslr. No. 481 (1967); BBA 242, 172 (1971).
10.
YEAST AND NEUROSPORA INVERTASES
297
4000 IU/mg protein, although individual preparations have exceeded 5000 IU. It should be noted that Neumann and Lampen (56)used heating and ethanol precipitation and regularly obtained apparently homogeneous material with a specific activity of about 3000 IU. After elimination of these steps, potencies of 4000 to 5000 IU have been routine. This suggests that a significant amount of inactivation occurs during these treatments and that the partially inactive material subsequently fractionates with the native enzyme. Many of the purified preparations (especially those in which the invertase was extracted rapidly rather than by prolonged autolysis) contain about 50% carbohydrate. Neumann and Lampen (36,&) found that the mannan is covalently attached to the protein moiety of the enzyme, probably through glucosaminyl-asparagine bonds. The carbohydrate of Berggren’s (7) purified invertase was not removed by dimeriaation with Hgz+ or by gel filtration in 4 M urea and 0.1 M mercaptoethanol ; the mannan and protein were considered to be covalently linked. Greiling et aZ. (43) reported that their invertase preparation contained mannan linked to serine or threonine by O-glycosidic bonds. Few, if any, such alkali-labile linkages were detected by Neumann and Lampen (46) although this type of linkage is common in the bulk structural mannan of the yeast wall (46). Since the enzyme studied by Greiling et aZ. (49) contained almost 80% mannan and no evidence of homogeneity was presented, the 0-glycosides may have been present in mannan peptides not linked to the invertase molecules. Invertase samples purified by procedures which include prolonged autolysis frequently contain low amounts of carbohydrate, especially if the enzyme has been collected by precipitation with ammonium sulfate (42, 47). [The carbohydrate-free internal enzyme is readily precipitated in 70% saturated ammonium sulfate (6) ; the external glycoprotein enzyme is relatively soluble even in saturated ammonium sulfate (36).] In one instance (48) the mannan could be removed from the preparation by treatment with bentonite a t pH 2.9 leaving an unstable enzyme; the carbohydrate-free internal invertase is also unstable a t this pH (18). The best working hypothesis is that external invertase is a glycoprotein containing about 50% mannan and 2 4 % glucosamine (probably the N-acetyl form). During prolonged autolysis much of the mannan can be removed, leaving a heterogeneous enzyme with a low or even negligible mannan content. 45. N. P. Neumann and J. 0. Lampen, Biochembtly 8, 3552 (1969). 46. R. Sentandreu and D. H. Northcote, BJ 109, 419 (1968). 47, M. Adams and C. S. Hudson, JAC8 65, 1359 (1943). 48. E. H. Fischer, L. Kohtes, and J. Fellig, Helv. Chim. Acta 34, 1132 (1951)
298
J. OLIVER LAMPEN
The carbohydrate moiety of external invertase is not essential for its catalytic activity since several highly active preparations have been almost devoid of carbohydrate (42,47, 48). Also, the internal invertase has a t least two-thirds the specific activity of the best external preparations [the value of this comparison is limited, however, since the amino acid compositions of the internal and external enzymes differ strikingly in the single strain examined (18)1. The carbohydrate moiety does increase thc stability of the external enzyme to a variety of agents. Arnold (49) has shown that susceptibility to heat inactivation decreases with increasing carbohydrate level. The internal (carbohydrate-free) enzyme is more sensitive to acid pH than is the external enzyme (18), although it is more stable a t alkaline pH. Also, the external enzyme is unaffected by condensed tannins, whereas the internal enzyme is sensitive (60).
D. PROPERTIES OF PURIFIED ENZYME Most of the external invertase preparations of high specific activity listed in Table I were reasonably homogeneous in that they gave a single band upon polyacrylamide gel electrophoresis and have usually shown single though broad peaks upon gel filtration or ion exchange chromatography. The enzyme from Saccharomyces mutant FH4C (56) is free of substantial contamination with nucleic acid (A2so:A260 = 1.8) and has an Eig (based on protein content) of 23.0. On ultracentrifugaof 10.4s (molecular weight tion it showed a major component with s20,w 270,000 i= 11,000 daltons) and a minor peak, probably a dimer, which mas also enzymically active. Material from the main peak did not aggregate when rerun under the same conditions; in contrast, the purified invertase from Saccharomyces strain LK2G12 aggregates readily and reversibly (S5). Although the specific activity per mg protein is reasonably uniform across tlic major pcak from ion excliangc chromatography (35),the carbohydrate content usually shifts, e.g., from 52 to 53% a t the front of the peak to 47-48% at the back. This variation may result from partial enzymic removal of the mannan even during rapid purification, or the size of the carbohydratc side chains may vary naturally. I n the one preparation examined (45) approximately 30 chains of mannan with a size range of 2,000-10,000 daltons were present per molecule of enzyme, linked to the protein through glucosaminyl-asparagine bonds. The 49. W. N. h n o l d . BBA 178, 247 (1969). 50. D. H. Strumeyer and M. J. Malin, BJ 118, 899 (1970).
10. YEAST
299
AND NEUROSPORA IKVERTASES
mannan associated with invertase has been reported (61) to be identical to the bulk mannan of the yeast wall, but the mannan of FH4C invertase (46) differs a t least in that it contains few, if any, of the serine- or threonine-0-mannose linkages characteristic of the wall mannan (43, 46). The amino acid compositions of external invertase from brewer's yeast (42) and mutant FH4C (18) and of the internal enzyme from the mutant (18) are compared in Table 11. The distribution of amino acids in the two external enzymes is similar and contrasts sharply with the internal enzyme, especially in the levels of glycine, tyrosine, histidine, and halfcystine. The external enzyme from mutant FH4C was not activated by cysteine (18) [although Hoshino and Momose (62)did report a fourfold TABLE I1 AMINOACIDCOMPOSITION OF PURIFIED INTERNAL AND EXTEBNAL INVERTABES Molee/135,000 g of protein
External Amino acid
[Ref. (IS)]
Glycine Alanine Serine Threonine Proline Valiie Isoleucine Leucine Phenylalanine Tyrosine Tryptophan Half-cystine Methionine Aspartic acid Glutamic acid Arginine Histidine Lysine Glucosamine
71 68 114 84 65 69 40 83 80 65 33 5 21 178 115 27
(Mannose)
(50%)
a
16
60 38
Internal [Ref. (U)] 74 72 129 106
115 84
a
151 80 63 73 38 77 77 31 30 0 14 165 124 32 29 85 0
(13%)
(<3%)
64 68
39 76
85 71 a
>1 19 171 116 27 16 54
Not determined.
51. A. J. Cifonelli and F. Smith, JACS 7'7, 5682 (1955). 52. J. Hoshino and A. Momose, J . Gen. A p p l . Microbid. 12, 163 (1966).
300
J . OLIVER LAMPEN
activation of two forms from baker’s yeast], nor was it sensitive to or reactive with sulfhydryl reagents (36).Three SH groups were accessible to iodoacetate in 8 M urea (35) ; the remaining two residues may exist as a disulfide bridge. The pH-stability curves for the external and internal forms differ markedly (18). The external enzyme is inactivated a t pH 8 and above, but it is stable at pH 3. The internal form is stable a t pH 9, but it undergoes reversible inactivation at pH 5 or less. During this inactivation there was no change in the molecular weight of the internal enzyme.
E. CATALYTIC PROPERTIW 1. Active Site
To be cleaved by yeast invertase a substrate must contain an unsubstituted P-D-fructofuranosyl residue (1, 3 ) . Gottschalk (3) has concluded that the enzyme combines primarily with this moiety through the glycosidic oxygen and the OH groups a t C-6 and C-3 of fructose. There may be some specific interaction with the OH a t C-2 of glucose since sucrose is the best substrate. Large “afructon” groups decrease the rate of cleavage, possibly through steric factors. Rupture of the glycosidic linkage occurs on the fructose side of the glycosidic oxygen (53, 6 4 ) . Binding of the substrate involves a group(s) with pK c 3 (55-57) which determines the acid side of the pH-activity curve. The most attractive possibility is a carboxylic group. The alkaline side of the pH-activity curve has the form of a dissociation curve of a weak acid with pK z 7, probably the imidazole group of a histidine residue ( I , 5 8 ) . This residue does not appear to be involved in substrate binding since the alkaline side of the pH curve is not dependent on substrate concentration (59) [such a dependence has clearly been demonstrated for the acid limb ( G O ) ] . Thus the active form of the enzyme requires at least an unfor substrate binding and a proprotonated acidic group (COO-?) tonated imidazole residue which is apparently involved in substrate cleavage. 53. D.E.Koshlnnd, Jr., and S. S. Stein, JBC 208, 139 (1954). 54. D.E. Koshland, Jr., Biol. Rev. 28, 416 (1953). 55. K. Myrback, 2. Physiol. Chem. 158, 160 (1926). 56. K. Myrback, SOC. Biol. Chemists, India, Silver Jubilee Souvenir p. 204 (1956). 57. K.Myrback and E. Willstaedt, Arkiv Kemi 15, 379 (1960). 58. S. Shall and A. Waheed. BJ 111, 33P (1969). 59. R. Kuhn, 2.Physiol. Chem. 125, 28 (1923). 60. K. Myrback and U. Bjorklund, Arkiv Kemi 4, 567 (1952).
10.
YEAST AND NEC'ROSPORA ISTERTASES
301
Sulfhydryl Groups. It has frequently been suggested that an SH group participates in the catalytic action of invertase. The activity of some preparations can be increased several fold by cysteine (529, and the enzyme can be inactivated by iodine and by Hg2+ or Ag2+ ( l ) ,although not by the more specific SH reagents (35). Also the iodine-invertase produced at pH 5.0 (which retains about 50% of the original activity) is much more resistant to Hg" o U g ' + tha9 is the native enzyme. This led to the suggestion that iodine reacts at pH 5.0 with an SH group that is partially masked in the native form of invertase (1). The concept has been questioned since cysteine, glutathione, awl ascorbic acid do not produce reactivation (61), but it was supported by the observation of Shall and Waheed (58) that nonacidic thiols (mercaptoethanol and mercaptoethylamine) were effective. The latter workers (58) proposed that lack of reactivation by cysteine resulted from charge repulsion by a COOgroup a t or near the active center. , The mechanism of iodine inactivation and indeed the question of any catalytic role for the SH groups of external invertase must, however, be left undecided in light of the fwt that internal invertase lacks cysteine residues yet shows catalytic properties) very similar to those of the external enzyme (18). (Recent studies by Q.Waheed in the author's laboratory have shown that internal invertase also reacts with iodine to yield a product with approximately half the original activity.) The most tenable assumption for the present is that iodine does not react with an SH of the external enzyme [nor with , a tyrosine or histidine residue (58)] but possibly oxidizes a methionine residue to the sulfoxide [cf. Koshland et al. (62, 6S)I. 2. Inhibitors
Inhibition of invertase by Znz+is reversible and noncompetitive, and it is strongly dependent on p H ( 6 4 ) . The reactive group on the protein appears to be the unprotonated form of the imidazole group determining the alkaline side of the pH-activity curve. Myrback (64) proposed that Zn2+ (and Hg2+)links two enzyme molebules to form a dimer. Dimerization by Hgz+ has been demonstrated by Berggren (Y), who attempted unsuccessfully to separate the enzyme protein and the mannan in this manner. 61. K. Myrback and E. Willstaedt, Arkiv Kemi 13, 179 (1957). 62. D. E. Koshland, Jr., D. H. Strumeyer, and W. J. Ray, Jr., Brookhaven Sump. Biol. 15, 101 (1962). 63. M . E. Koshland, F. M. Englberger, M. J. Erwin, and S. M. Gaddone, JBC 238, 1343 (1963). 64. K. Myrback, Arkiv Kemi 27, 507 (1967).
302
J . OLIVER LAMPEN
Pyridoxal and pyridoxal phosphate are potent inhibitors of potato invertase (66), but they are much less effective against the yeast or Neurospora enzymes. I n contrast, the fungal enzymes are very sensitive to inhibition by aniline whereas the potato enzyme is resistant (65, 6 6 ) . Invertase undergoes i,mmediate reversible inactivation by concentrations of urea which do not produce major changes in physical properties (67). It appears to combine with ca. 1.3 molecules of urea per catalytic site (68). At high urea concentrations, e.g., 8 M at p H 5.0, or a t pH values above 6.0, irreversible inactivation occurs along with changes in secondary and tertiary structure. 3. Kinetics
The values of K , for soluble invertase and for the enzyme bound to cell walls were identical [determined from plots of the inhibition by aniline ( 6 9 ) ] .The K , for the wall preparations was lower (33 mM) than for the soluble enzyme (44 mM) ; this was considered to reflect some hindrance imposed by the site of the bound enzyme. Invertase insolubilized by ionic binding to DEAE-cellulose was prepared by Suzuki e t nl. (70). The apparent pH optimum was displaced from pH 5.4 for the free enzyme to pH 3.4 for the bound; however, the latter value is probably not a true optimum pH since the DEAE-cellulose would probably provide a microenvironment of higher pH than that of the external buffer. 4. Mechanism
Following discovery of the activity of yeast invertase in transferring P-fructofuranosyl units to primary alcohols [for references, cf. Myrbiick ( I ) ] , it has frequently been proposed that a two-step reaction occurs with an active enzyme-fructose complex as the first step. This can be written as IEH+GOFsEF+GOH I1 EF HOH s EH FOH I11 EF XOH e EH XOF
+ +
+ +
65. R. Pressey, BBA 159, 414 (1968). 66. K. Myrbiick, 2. Physiol. Chem. 158, 160 (1926). 67. A. M. Chase and M. S. Krotkov, J . Cellular Comp. Physiol. 47, 305 (1956). 68. A. M. Chase, H. C. von Meier, and V. J. Menna, J . Cellular Comp. Physiol. 59, 1 (1962). 69. M. V. Tracey, BBA 77, 147 (1963). 70. H. Sueuki, Y. Oeawa, H. Maeda, and 0. Tanabe, R e p t . Ferment. Res. Inst. 31, 11 (1967).
10.
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where E stands for enzyme, GOF for sucrose, GOH and FOH for glucose and fructose, and XOH any receptor sugar. Andersen (71) has recently shown that high concentrations of free fructose (but not glucose) can act as both donor and acceptor giving rise to fructose disaccharides. This provides strong support for the existence of an active enzyme-fructose complex. The qualitative and quantitative distribution of mono-, di-, tri-, and tetrasaccharides during sucrose cleavage is also consistent with a twostep reaction mechanism in which the first step is the formation of an enzyme-fructose complex (72). 111. Neurospora Invertare
The invertase of N . crussa has many features in common with that from Saccharomyces yeasts. Most of the enzyme is retained by the fungal cell and is external to the cell membrane; thus, it is freely accessible to exogenous substrates and has kinetic parameters and sensitivity t o inhibitors identical with those of the free enzyme (73). By a variety of biochemical and histochemical proceduies, the cell-bound enzyme has been shown to be partly in the cell wall and partly between the wall and the cell membrane (73-76). A portion of the enzyme is bound very tightly by the wall and can be extracted only with difficulty (73, 75). Two sizes of active enzyme are present in Neurospora (77, 78), and there is a preferential release of the smaller form during growth (79). Since Manocha and Colvin (80) reported the occurrence of discrete pores in the cell wall of Neurospora, it has been suggested that release of the external enzyme occurs through these pores with molecular sieving favoring the passage of the smaller form. The two sizes of Neurospora invertase are not differentially distributed as are those of yeast. Heavy (H) enzyme predominates both inside Neurospora protoplasts and in the walls of growing cells, but the light 71. B. Andersen, Acta Chem. Scand. 21, 828'(1967). 72. B. Andersen, N. Thiesen, and P. E. Broe, Acta Chem. Scand. 23, 2367 (1969). 73. R. L. Metsenberg, BBA 74, 455 (1963). 74. E. P. Hill and A. S. Sussman, J . Bacteriol. 88, 1556 (1964). 75. M. L. Sargent and D. 0. Woodward, J . Bacteriol. 97, 867 (1969). 76. P. L. Y. Chung and J. R. Trevithick, J . Bacte+l. 102, 423 (1970). 77. F. I. Eilers, J. Allen, E. P. Hill, and A. S. Sussman, J . Hislochem. Cytochem. 12, 448 (1964). 78. R. L. Metsenberg, ABB 100, 503 (1963). 79. J. R. Trevithick and R. L. Metsenberg, J . Bacteriol. 92, 1010 and 1016 (1968). 80. M. Manocha and J. Colvin, J . Bacten'ol. 94, 202 (1967).
304
J. OLIVEB LAMPEN
(L) enzyme is always present in small amounts (81, 82). The L enzyme is favored at low pH and can be produced from the H form by heating or by high salt concentration either a t acid or alkaline pH (82). It reaggregates in concentrated solutions in the absence of salt (e.g., during dialysis of an ammonium sulfate precipitate). A. PURIFIED PREPARATIONS Neurospora crassa invertase was first obtained in a homogeneous state by Metzenberg (78) who purified the material extractable from mycelial powder by pH 5.0 buffer. The preparation was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. It contained 2.4% hexosamine but no detectable neutral sugar. Shortly thereafter Eilers et al. (77) noted that crude extracts of N . crassa gave two bands of activity by gel electrophoresis. This observation was confirmed by Metzenberg (82) and the two components separated by gel filtration into heavy (H) and light (L) forms. Typically 65435% of the activity was in the H form. The szo,wvalues for the H and L enzymes were 10.3 and 5.2, respectively. As already noted the H form can be converted to L enzyme by heat or high salt. During dissociation a transient form with s20,w= 9.8 could be detected. This was considered to be the polymeric enzyme in a preclastic conformation (82). From Metzenberg’s data it can be suggested that Neurospora invertase occurs primarily as the polymeric H form which dissociates under appropriate conditions to yield four active subunits (if both molecules are assumed to be spherical). No evidence was obtained for more than one type of subunit (82). The L enzyme obtained by dissociation of the H enzyme seems to be identical to that present in cell extracts. Dissociation in vitro caused 31% loss of activity; but there was no change of activity during reaggregation, so that the true activity of the subunits may be the same in monomeric and in polymeric form. Solutions of L invertasc foamed copiously upon shaking, whereas H invertase showed little tendency to foam (82). This was considered to indicate that the hydrophobic side chains of H invertase are oriented inwardly and become exposed during dissociation. According to this concept hydrophobic bondings might serve to maintain the polymeric structure of the H form. Pure invertase has recently been isolated from another high-producing strain of N . crassa (37) by Meachum et al. (83).The striking charac81. J. R. Trevithick and R . L. Metzenberg, BBRC 16, 319 (1964). 82. R. L. Metzenberg, BBA 89, 291 (1964). 83. Z. D. Meachum, Jr., H. J. Colvin, Jr., and H. D. Braymer, Biochemistry 10, 326 (1971).
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AND NEUROSPORA INVERTASES
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teristic of this material is that it contains approximately 11% mannose and 3% glucosamine covalently linked to the protein moiety. Proteinase digestion yielded peptides containing glucosamine and aspartic acid, suggesting that the glucosaminyl-asparagine bond predominates here as in yeast external invertase (45).If serine-0-glycoside linkages are present, their number must be small. The isolated enzyme had an s,O,,, of 10.5 and a molecular weight of 210,000. Four disulfide bonds were detected per mole. In the presence of a dissociating agent (6 M guanidine) and under reducing conditions, the s20,wwas 5.2 and the molecular weight 51,500.The native enzyme thus appears to be a tetramer and, from tryptic peptide fingerprints, probably contains more than one type of subunit. As with Metzenberg's strain (82) both the 10.5 S and 5.2 S forms are found in crude cell extracts and both are enzymically active. The specific activity of Braymer's (83) purified enzyme is 1820 pmoles of sucrose split per minute per milligram of glycoprotein; the value for Metzenberg's material (78) is 1890 poles/minute.
B . CATALYTIC PROPERTIES The substrate specificity of Neurospora invertase (78) is similar to that of the yeast enzyme. Sucrose is the most readily utilized substrate with a K,, of 6.1 x 10-3M and the highest VMx. For raffinose, K,,, is 6.5 x W S M and the V , , , 20% of that for sucrose. The values for &methyl fructoside are K , of 3.3 x lo-* M and relative V,,, of 30%. [These K , values are notably lower than the 26 X M for sucrose and 150 x M for raffinose obtained with the purified yeast enzyme (18).] Trehalose and melezitose were not attacked. The activation energies (calculated from rates a t 0" and 38") are 10.8, 12.3, and 13.9 kcal for sucrose, raffinose, and P-methyl fructoside, respectively. The pH-activity curve has a broad optimum at pH 4.5-6.0. The alkaline limb approximates that for yeast invertase, but the acid side falls off rapidly below pH 4.5 (78). Neurospora invertase is less sensitive than the yeast enzyme to inhibition by divalent cations. Zn2+and Cu2+caused only 33 and 79% inhibition, respectively, a t 0.05M . Both enzymes are strongly inhibited by aniline (65, 78). The sulfhydryl reagent p-hydroxymercuribenzoate inhibits the fungal enzyme and partial protection is afforded by the presence of substrate (sucrose). In contrast the purified yeast external enzyme was not inhibited by several sulfhydryl reagents, including p-mercuribenzoate (35).