Mutations affecting the activity of the cyclodextrin glucanotransferase of Paenibacillus pabuli US132: insights into the low hydrolytic activity of cyclodextrin glucanotransferases

The cyclodextrin glucanotransferase from Paenibacillus pabuli US132 (US132 CGTase) was engineered using a rational approach in an attempt to provide it with anti-staling properties comparable to those of the commercial maltogenic amylase (Novamyl). The study aimed to concurrently decrease the cyclization activity and increase the hydrolytic activity of US132 CGTase. A five-residue loop (PAGFS) was inserted, alone or with the substitution of essential residues for cyclization (G180, L194 and Y195), mimicking the case of Novamyl. The findings indicate that, unlike the case of the CGTase of Thermoanerobacterium thermosulfurigenes strain EM1 whose initial high hydrolytic activity was exceptional, these mutations completely abolished the cyclization and hydrolytic activities of the US132 CGTase. This suggests that those mutations are not able to convert conventional CGTases, whose hydrolytic activities are very weak, into hydrolases. Accordingly, and for the first time, a structural barrier at subsite ?3 was advanced as an influential factor which might explain the low hydrolytic activity of conventional CGTases.     

The residue 179 is involved in product specificity of the <Emphasis Type="Italic">Bacillus circulans</Emphasis> DF 9R cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferases (CGTases) are important enzymes in biotechnology because of their ability to produce cyclodextrin (CD) mixtures from starch whose relative composition depends on enzyme source. A multiple alignment of 46 CGTases and Shannon entropy analysis allowed us to find differences and similarities that could be related to product specificity. Interestingly, position 179 has Gly in all the CGTases except in that from Bacillus circulans DF 9R which possesses Gln. The absence of a side chain at that position has been considered as a strong requirement for substrate binding and cyclization process. Therefore, we constructed two mutants of this enzyme, Q179L and Q179G. The activity and kinetic parameters of Q179G remained unchanged while the Q179L mutant showed a different CDs ratio, a lower catalytic efficiency, and a decreased ability to convert starch into CDs. We show that position 179 is involved in CGTase product specificity and must be occupied by Gly—without a side chain—or by amino acid residues able to interact with the substrate through hydrogen bonds in a way that the cyclization process occurs efficiently. These findings are also explained on the basis of a structural model.     

Enzymatic production of cyclodextrins

Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly alpha-amylases. Due to its low solubility in water, separation and purification of beta-CD is relatively easy compared to alpha- and gamma-CD. In recent years more economic processes for gamma-CD and especially alpha-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of alpha-CD production processes is presented.     

Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and cyclodextrin size specificity

The enzymes from the alpha-amylase family all share a similar alpha-retaining catalytic mechanism but can have different reaction and product specificities. One family member, cyclodextrin glycosyltransferase (CGTase), has an uncommonly high transglycosylation activity and is able to form cyclodextrins. We have determined the 2.0 and 2.5 A X-ray structures of E257A/D229A CGTase in complex with maltoheptaose and maltohexaose. Both sugars are bound at the donor subsites of the active site and the acceptor subsites are empty. These structures mimic a reaction stage in which a covalent enzyme-sugar intermediate awaits binding of an acceptor molecule. Comparison of these structures with CGTase-substrate and CGTase-product complexes reveals three different conformational states for the CGTase active site that are characterized by different orientations of the centrally located residue Tyr 195. In the maltoheptaose and maltohexaose-complexed conformation, CGTase hinders binding of an acceptor sugar at subsite +1, which suggests an induced-fit mechanism that could explain the transglycosylation activity of CGTase. In addition, the maltoheptaose and maltohexaose complexes give insight into the cyclodextrin size specificity of CGTases, since they precede alpha-cyclodextrin (six glucoses) and beta-cyclodextrin (seven glucoses) formation, respectively. Both ligands show conformational differences at specific sugar binding subsites, suggesting that these determine cyclodextrin product size specificity, which is confirmed by site-directed mutagenesis experiments.     

The structure of Pseudomonas P51 Cl-muconate lactonizing enzyme: co-evolution of structure and dynamics with the dehalogenation function

Bacterial muconate lactonizing enzymes (MLEs) catalyze the conversion of cis,cis-muconate as a part of the beta-ketoadipate pathway, and some MLEs are also able to dehalogenate chlorinated muconates (Cl-MLEs). The basis for the Cl-MLEs dehalogenating activity is still unclear. To further elucidate the differences between MLEs and Cl-MLEs, we have solved the structure of Pseudomonas P51 Cl-MLE at 1.95 A resolution. Comparison of Pseudomonas MLE and Cl-MLE structures reveals the presence of a large cavity in the Cl-MLEs. The cavity may be related to conformational changes on substrate binding in Cl-MLEs, at Gly52. Site-directed mutagenesis on Pseudomonas MLE core positions to the equivalent Cl-MLE residues showed that the variant Thr52Gly was rather inactive, whereas the Thr52Gly-Phe103Ser variant had regained part of the activity. These residues form a hydrogen bond in the Cl-MLEs. The Cl-MLE structure, as a result of the Thr-to-Gly change, is more flexible than MLE: As a mobile loop closes over the active site, a conformational change at Gly52 is observed in Cl-MLEs. The loose packing and structural motions in Cl-MLE may be required for the rotation of the lactone ring in the active site necessary for the dehalogenating activity of Cl-MLEs. Furthermore, we also suggest that differences in the active site mobile loop sequence between MLEs and Cl-MLEs result in lower active site polarity in Cl-MLEs, possibly affecting catalysis. These changes could result in slower product release from Cl-MLEs and make it a better enzyme for dehalogenation of substrate.     

X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7A resolution.

The three-dimensional structure of the Bacillus stearothermophilus "maltogenic" alpha-amylase, Novamyl, has been determined by X-ray crystallography at a resolution of 1.7 A. Unlike conventional alpha-amylases from glycoside hydrolase family 13, Novamyl exhibits the five-domain structure more usually associated with cyclodextrin glycosyltransferase. Complexes of the enzyme with both maltose and the inhibitor acarbose have been characterized. In the maltose complex, two molecules of maltose are found in the -1 to -2 and +2 to +3 subsites of the active site, with two more on the C and E domains. The C-domain maltose occupies a position identical to one previously observed in the Bacillus circulans CGTase structure [Lawson, C. L., et al. (1994) J. Mol. Biol. 236, 590-600], suggesting that the C-domain plays a genuine biological role in saccharide binding. In the acarbose-maltose complex, the tetrasaccharide inhibitor acarbose is found as an extended hexasaccharide species, bound in the -3 to +3 subsites. The transition state mimicking pseudosaccharide is bound in the -1 subsite of the enzyme in a 2H3 half-chair conformation, as expected. The active site of Novamyl lies in an open gully, fully consistent with its ability to perform internal cleavage via an endo as opposed to an exo activity.     

Catalytic function and affinity purification of site-directed mutant β-cyclodextrin glucanotransferase from alkalophilic Bacillus firmus var. alkalophilus

Subsites −3 and −7 in the active site of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilic Bacillus firmus var. alkalophilus were modified through site-directed mutagenesis to obtain novel mutant CGTases. Four mutant CGTases, H59Q, Y96M, 90-PPI-92, and Δ(154–160) were constructed and produced using a recombinant E. coli with a secretive expression system extracellularly. The secreted mutant β-CGTases were purified by one-step affinity adsorption chromatography using a β-cyclodextrin (CD) polymer as an adsorbent to nearly homogeneous purity. The catalytic activities were modified significantly compared to the wild-type. In particular, the Y96M and Δ(154–160) mutants increased cyclization activity around 1.5 times without any significant reduction of coupling and hydrolyzing activities. Meanwhile, the Y96M and Δ(154–160) mutants exhibited a much higher conversion yield into CDs from 28.6 to 39% without any recognizable change in the CD ratio. The conversion yield into linear maltooligosaccharides was also significantly reduced. The catalytic functions of subsites −3 and −7 in the active site of β-CGTase would appear to be related to the overall productivity of CDs rather than the product specificity.     

Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production

Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.     

Kinetic and structural analysis of mutant Escherichia coli dihydroorotases: a flexible loop stabilizes the transition state

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-l-aspartate (CA-asp) to l-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. Two different conformations of the surface loop (residues 105-115) were found in the dimeric Escherichia coli DHOase crystallized in the presence of DHO (PDB code 1XGE). The loop asymmetry reflected that of the active site contents of the two subunits: the product, DHO, was bound in the active site of one subunit and the substrate, CA-asp, in the active site of the other. In the substrate- (CA-asp-) bound subunit, the surface loop reaches in toward the active site and makes hydrogen bonds with the bound CA-asp via two threonine residues (Thr109 and Thr110), whereas the loop forms part of the surface of the protein in the product- (DHO-) bound subunit. To investigate the relationship between the structural states of this loop and the catalytic mechanism of the enzyme, a series of mutant DHOases including deletion of the flexible loop were generated and characterized kinetically and structurally. Disruption of the hydrogen bonds between the surface loop and the substrate results in significant loss of catalytic activity. Furthermore, structures of these mutants with low catalytic activity have no interpretable electron density for parts of the flexible loop. The structure of the mutant (Delta107-116), in which the flexible loop is deleted, shows only small differences in positions of other substrate binding residues and in the binuclear zinc center compared with the native structure, yet the enzyme has negligible activity. The kinetic and structural analyses suggest that Thr109 and Thr110 in the flexible loop provide productive binding of substrate and stabilize the transition-state intermediate, thereby increasing catalytic activity.     

Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step. Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP. Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA. Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA. These results extend our recent modeling studies, which predicted that the residues of the active center cleft could, via electrostatic interactions, contribute to both the correct orientation and stable binding of the substrate RNA molecules in PAP active site pocket. The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.     

Soluble aldose sugar dehydrogenase from Escherichia coli: a highly exposed active site conferring broad substrate specificity

A water-soluble aldose sugar dehydrogenase (Asd) has been purified for the first time from Escherichia coli. The enzyme is able to act upon a broad range of aldose sugars, encompassing hexoses, pentoses, disaccharides, and trisaccharides, and is able to oxidize glucose to gluconolactone with subsequent hydrolysis to gluconic acid. The enzyme shows the ability to bind pyrroloquinoline quinone (PQQ) in the presence of Ca2+ in a manner that is proportional to its catalytic activity. The x-ray structure has been determined in the apo-form and as the PQQ-bound active holoenzyme. The beta-propeller fold of this protein is conserved between E. coli Asd and Acinetobacter calcoaceticus soluble glucose dehydrogenase (sGdh), with major structural differences lying in loop and surface-exposed regions. Many of the residues involved in binding the cofactor are conserved between the two enzymes, but significant differences exist in residues likely to contact substrates. PQQ is bound in a large cleft in the protein surface and is uniquely solvent-accessible compared with other PQQ enzymes. The exposed and charged nature of the active site and the activity profile of this enzyme indicate possible factors that underlie a low affinity for glucose but generic broad substrate specificity for aldose sugars. These structural and catalytic properties of the enzymes have led us to propose that E. coli Asd provides a prototype structure for a new subgroup of PQQ-dependent soluble dehydrogenases that is distinct from the A. calcoaceticus sGdh subgroup.     

Randomization of the receptor alpha chain recruitment epitope reveals a functional interleukin-5 with charge depletion in the CD loop.

We report the functional phage display of single chain human interleukin-5 (scIL-5) and its use for receptor-binding epitope randomization. Enzyme-linked immunosorbent assays and optical biosensor analyses verified expression of scIL-5 on the phage surface and binding of scIL-5 phage to interleukin-5 receptor alpha chain. Furthermore, an asymmetrically disabled but functional scIL-5 mutant, (wt/A5)scIL-5, was displayed on phage. (wt/A5)scIL-5 was constructed from an N-terminal half containing the original five charged residues (88EERRR92) in the CD loop, including the Glu89 and Arg91 believed key in the alpha chain recognition site, combined with a C-terminal half containing a disabled CD loop sequence (88AAAAA92) missing the key recognition residues. This asymmetric variant was used as a starting point to generate an scIL-5 library in which the intact 88-92 N-terminal CD loop was randomized. From this epitope library, a receptor-binding variant of IL-5 was detected, (SLRGG/A5)scIL-5, in which the only charged residue in the CD loop is an Arg at position 90. Characterization of this variant expressed as a soluble protein in E. coli shows that the IL-5 pharmacophore for receptor alpha chain binding can function with a single positive charge in the CD loop. Charge-depleted CD loop mimetics of IL-5 suggest the importance of charge distribution in functional IL-5 receptor recruitment.     

Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant

Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 A crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax.     

Modeling and alanine scanning mutagenesis studies of recombinant pokeweed antiviral protein

The Phytolacca americana-derived naturally occurring ribosome inhibitory protein pokeweed antiviral protein (PAP) is an N-glycosidase that catalytically removes a specific adenine residue from the stem loop of ribosomal RNA. We have employed molecular modeling studies using a novel model of PAP-RNA complexes and site-directed mutagenesis combined with bioassays to evaluate the importance of the residues at the catalytic site and a putative RNA binding active center cleft between the catalytic site and C-terminal domain for the enzymatic deadenylation of ribosomal RNA by PAP. As anticipated, alanine substitutions by site-directed mutagenesis of the PAP active site residues Tyr(72), Tyr(123), Glu(176), and Arg(179) that directly participate in the catalytic deadenylation of RNA resulted in greater than 3 logs of loss in depurinating and ribosome inhibitory activity. Similarly, alanine substitution of the conserved active site residue Trp(208), which results in the loss of stabilizing hydrophobic interactions with the ribose as well as a hydrogen bond to the phosphate backbone of the RNA substrate, caused greater than 3 logs of loss in enzymatic activity. By comparison, alanine substitutions of residues (28)KD(29), (80)FE(81), (111)SR(112), (166)FL(167) that are distant from the active site did not significantly reduce the enzymatic activity of PAP. Our modeling studies predicted that the residues of the active center cleft could via electrostatic interactions contribute to both the correct orientation and stable binding of the substrate RNA molecule in the active site pocket. Notably, alanine substitutions of the highly conserved, charged, and polar residues of the active site cleft including (48)KY(49), (67)RR(68), (69)NN(70), and (90)FND(92) substantially reduced the depurinating and ribosome inhibitory activity of PAP. These results provide unprecedented evidence that besides the active site residues of PAP, the conserved, charged, and polar side chains located at its active center cleft also play a critical role in the PAP-mediated depurination of ribosomal RNA.     

Site-directed mutations in Alanine 223 and Glycine 255 in the acceptor site of gamma-Cyclodextrin glucanotransferase from Alkalophilic Bacillus clarkii 7364 affect cyclodextrin production

A cyclodextrin glucanotransferase (CGTase) from Bacillus clarkii 7364 converts starch into gamma-cyclodextrin (gamma-CD) with high specificity. Comparison of the deduced amino acid sequence of this CGTase with those of other typical CGTases revealed that several amino acids are deleted or substituted with others at several subsites. Of these amino acids, Ala223 at subsite +2 and Gly255 at subsite +3 in the acceptor site of the enzyme were replaced by several amino acids through site-directed mutagenesis. The replacement of Ala223 by lysine, arginine and histidine strongly enhanced the gamma-CD-forming activity in the neutral pH range. On the other hand, all mutants obtained on replacing Gly255 with the above amino acids showed significant decreases in the gamma-CD-forming activity. Taking into account both the kinetic parameters and pKa values of the side chains of the three basic amino acids, the protonation state of the amino groups in their side chains at subsite +2 seems to enhance the hydrogen bonding interaction between these basic amino acids and the glucose residues of linear oligosaccharides. The enhancement of the interaction may play an important role by helping the substrate reach subsite +1, hence increasing the gamma-CD-forming activity and kcat value.     

Cloning and nucleotide sequence of a thermostable cyclodextrin glycosyltransferase gene from Thermoanaerobactersp. ATCC 53627 and its expression in Escherichia coli

A gene, cgtA, encoding an extremely thermostable cyclodextrin glycosyltransferase (CGTase) was cloned from a thermophilic anaerobe, Thermoanaerobacter sp. ATCC 53627, and expressed in Escherichia coli. DNA and protein sequencing revealed that the mature enzyme of 683 amino acid residues (MW 75 kDa) was preceded by a signal peptide of 27 amino acid residues. The sequence of the Thermoanaerobacter CGTase was similar to sequences of Bacillus CGTases, with more than 58% identity, and very similar (89% identity) to a CGTase enzyme from Thermoanaerobacterium thermosulfurogenes.     

Functional implications of the unstructured loop in the (beta/alpha)(8) barrel structure of the bacterial luciferase alpha subunit.

Bacterial luciferase catalyzes the conversion of FMNH(2), a long-chain aliphatic aldehyde, and molecular oxygen to FMN, the corresponding carboxylic acid, and H(2)O with the emission of light. The light-emitting species is an enzyme-bound excited state flavin. The enzyme is a heterodimer (alphabeta) of homologous subunits each with an (beta/alpha)(8) barrel structure. A portion of the loop in the alpha subunit that connects beta strand 7 to alpha helix 7 is disordered in the crystal structure. To test the hypothesis that this loop closes over the active site during catalysis and protects the active site from bulk solvent, a mutant was constructed in which the 29 residues that are disordered in the 2.4 A crystal structure were deleted. Deletion of this loop results in a heterodimer with a subunit equilibrium dissociation constant of 1.32 +/- 1.25 microM, whereas the wild-type heterodimer shows no measurable subunit dissociation. This mutant retains its ability to bind substrate flavin and aldehyde with wild-type affinity and can carry out the chemistry of the bioluminescence reaction with nearly wild-type efficiency. However, the bioluminescent quantum yield of the reaction is reduced nearly 2 orders of magnitude from that of the wild-type enzyme.     

Tomato pectin methylesterase: modeling, fluorescence, and inhibitor interaction studies-comparison with the bacterial (Erwinia chrysanthemi) enzyme

The molecular model of Lycopersicon esculentum (tomato) pectin methylesterase (PME) was built by using the X-ray crystal structure of PME from the phytopathogenic bacterium Erwinia chrysanthemi as a template. The overall structure and the position of catalytically important residues (Asp132, Asp 153, and Arg 221, located at the bottom of the active site cleft) are conserved. Instead, loop regions forming the walls of the catalytic site are much shorter and form a less deep cleft, as already revealed by the carrot PME crystal structure. The protein inhibitor of pectin methylesterase (PMEI) isolated from kiwi fruit binds tomato PME with high affinity. Conversely, no complex formation between the inhibitor and PME from E. chrysanthemi is observed, and the activity of this enzyme is unaffected by the presence of the inhibitor. Fluorescence quenching experiments on tomato PME and on PME-PMEI complex suggest that tryptophanyl residues present in the active site region are involved in the interaction and that the inhibitor interacts with plant PME at the level of the active site. We also suggest that the more open active site cleft of tomato PME allows the interaction with the inhibitor. Conversely, the narrow and deep cleft of the active site of E. chrysanthemi PME hinders this interaction. The pH-dependent changes in fluorescence emission intensity observed in tomato PME could arise as the result of protonation of an Asp residue with unusually high pKa, thus supporting the hypothesis that Asp132 acts as acid/base in the catalytic cycle.     

Crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 determined at 1.9 A resolution

The crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 was determined at 1.9 A resolution. While the wild-type CGTase from the same bacterium produces a mixture of mainly alpha-, beta- and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains, site-directed mutation of histidine-233 to asparagine changed the nature of the enzyme such that it no longer produced alpha-cyclodextrin. This is a promising step towards an industrial requirement, i.e. unification of the products from the enzyme. Two independent molecules were found in an asymmetric unit, related by pseudo two-fold symmetry. The backbone structure of the mutant enzyme was very similar to that of the wild-type CGTase except that the position of the side chain of residue 233 was such that it is not likely to participate in the catalytic function. The active site cleft was filled with several water molecules, forming a hydrogen bond network with various polar side chains of the enzyme, but not with asparagine-233. The differences in hydrogen bonds in the neighborhood of asparagine-233, maintaining the architecture of the active site cleft, seem to be responsible for the change in molecular recognition of both substrate and product of the mutant CGTase.     

The low-temperature luminescence properties of bovine alpha-lactalbumin.

The luminescence of bovine alpha-lactalbumin at 77 K has been studied and compared with that of lysozyme. Alpha-Lactalbumin has several unusual properties, including a fluorescence spectrum showing vibrational fine structure, an abnormal phosphorescence spectrum, a high fluorescence: phosphorescence ratio and an abnormal phosphorescence decay. These properties are largely due to the proximity of tryptophan residues to disulphide bonds. Reduction of all these bonds causes considerable changes in alpha-lactalbumin luminescence, as does denaturation in acid solution. Reduction of a single labile disulphide bond has little effect, and the properties of alpha-lactalbumin III, a variant lacking one disulphide bond and one trypotophan residue, are similar to those of the normal protein. Several differences between alpha-lactalbumin and lysozyme are reported. The results support the suggestion that the two tryptophan residues found in the active site cleft of alpha-lactalbumin may be largely responsible for its luminescence.