Site-directed mutational analysis of the novel catalytic domains of <Emphasis Type="Italic">α</Emphasis>-aminoadipate reductase (Lys2p) from <Emphasis Type="Italic"> Candida albicans</Emphasis>

The alpha-aminoadipate reductase, a novel enzyme in the alpha-aminoadipic acid pathway for the biosynthesis of lysine in fungi, catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde in the presence of ATP, NADPH and MgCl(2). This reaction requires two distinct gene products, Lys2p and Lys5p. In the presence of CoA, Lys5p posttranslationally activates Lys2p for the alpha-aminoadipate reductase activity. Sequence alignments indicate the presence of all functional domains required for the activation, adenylation, dehydrogenation and alpha-aminoadipic acid binding in the Lys2p. In this report we present the results of site-directed mutational analysis of the conserved amino acid residues in the catalytic domains of Lys2p from the pathogenic yeast Candida albicans. Mutants were generated in the LYS2 sequence of pCaLYS2SEI by PCR mutagenesis and expressed in E. coli BL21 cells. Recombinant mutants and the wild-type Lys2p were analyzed for their alpha-aminoadipate reductase activity. Substitution of threonine 416, glycine 418, serine 419, and lysine 424 of the adenylation domain (TXGSXXXXK, residues 416-424) resulted in a significant reduction in alpha-aminoadipate reductase activity compared to the unmutagenized Lys2p control. Similarly replacement of glycine 978, threonine 980, glycine 981, phenylalanine 982, leucine 983 and glycine 984 of the NADPH binding domain (GXTGFLG, residues 978-984) caused a drastic decrease in alpha-aminoadipate reductase activity. Finally, substitution of histidine 460, aspartic acid 461, proline 462, isoleucine 463, glutamine 464, arginine 465, and aspartic acid 466 of the putative alpha-aminoadipic acid binding domain (HDPIQRD, residues 460-466) resulted in a highly reduced alpha-aminoadipate reductase activity. These results confirm the hypothesis that specific amino acid residues in highly conserved catalytic domains of Lys2p are essential for the alpha-aminoadipate reductase activity.     

Lysine biosynthesis in Penicillium chrysogenum is regulated by feedback inhibition of α-aminoadipate reductase

A partially purified preparation of alpha-aminoadipate reductase (EC 1.2.1.31) from Penicillium chrysogenum is competitively inhibited by lysine (Ki of 0.26 mM). Exogenous addition of 10 mM L-lysine to resting mycelia of P. chrysogenum increased the intracellular lysine pool concentration 2-fold, but decreased the incorporation of (6-14C)-alpha-aminoadipate into protein-bound lysine to a fifth. The distribution of radioactivity in the pathway metabolites alpha-aminoadipate, saccharopine and lysine was consistent with the assumption of a lysine sensitive enzyme step in vivo between alpha-aminoadipate and saccharopine. Hence lysine inhibition of alpha-aminoadipate reductase may be of physiologic importance.     

Lysine biosynthesis in selected pathogenic fungi: characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans.

The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. Until now, this unique metabolic pathway has never been investigated in the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. Five of the eight enzymes (homocitrate synthase, homoisocitrate dehydrogenase, alpha-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase) of the alpha-aminoadipate pathway and glucose-6-phosphate dehydrogenase, a glycolytic enzyme used as a control, were demonstrated in wild-type cells of these organisms. All enzymes were present in Saccharomyces cerevisiae and the pathogenic organisms except C. neoformans 32608 serotype C, which exhibited no saccharopine reductase activity. The levels of enzyme activity varied considerably from strain to strain. Variation among organisms was also observed for the control enzyme. Among the pathogens, C. albicans exhibited much higher homocitrate synthase, homoisocitrate dehydrogenase, and alpha-aminoadipate reductase activities. Seven lysine auxotrophs of C. albicans and one of Candida tropicalis were characterized biochemically to determine the biochemical blocks and gene-enzyme relationships. Growth responses to alpha-aminoadipate- and lysine-supplemented media, accumulation of alpha-aminoadipate semialdehyde, and the lack of enzyme activity revealed that five of the mutants (WA104, WA153, WC7-1-3, WD1-31-2, and A5155) were blocked at the alpha-aminoadipate reductase step, two (STN57 and WD1-3-6) were blocked at the saccharopine dehydrogenase step, and the C. tropicalis mutant (X-16) was blocked at the saccharopine reductase step. The cloned LYS1 gene of C. albicans in the recombinant plasmid YpB1078 complemented saccharopine dehydrogenase (lys1) mutants of S. cerevisiae and C. albicans. The Lys1+ transformed strains exhibited significant saccharopine dehydrogenase activity in comparison with untransformed mutants. The cloned LYS1 gene has been localized on a 1.8-kb HindIII DNA insert of the recombinant plasmid YpB1041RG1. These results established the gene-enzyme relationship in the second half of the alpha-aminoadipate pathway. The presence of this unique pathway in the pathogenic fungi could be useful for their rapid detection and control.     

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.     

Crystal structure of the his-tagged saccharopine reductase from Saccharomyces cerevisiae at 1.7-Å resolution

The three-dimensional structure of the saccharopine reductase enzyme from the budding yeast Saccharomyces cerevisiae was determined to 1.7-A resolution in the apo form by using molecular replacement. The enzyme monomer consists of three domains: domain I is a variant of the Rossmann fold, domain II folds into a alpha/beta structure containing a mixed seven-stranded beta-sheet as the central core, and domain III has an all-helical fold. Comparative fold alignment with the enzyme from Magnaporthe grisea suggests that domain I binds to NADPH, and domain II binds to saccharopine and is involved in dimer formation. Domain III is involved in closing the active site of the enzyme once substrates are bound. Structural comparison of the saccharopine reductase enzymes from S. cerevisiae and M. grisea indicates that domain II has the highest number of conserved residues, suggesting that it plays an important role in substrate binding and in spatially orienting domains I and III.     

Biosynthesis of lysine in Rhodotorula glutinis: role of pipecolic acid.

Glutamate-alpha-ketoadipate transaminase, saccharopine reductase, and saccharopine dehydrogenase activities were demonstrated in extracts of Rhodotorula glutinis but alpha-aminoadipate reductase activity could not be measured in whole cells or in extracts. Lysine auxotroph lys1 grew in the presence of L-lysine or DL-alpha-aminoadipate and incorporated radioactivity from DL-alpha-amino-[I-14C]adipate into lysine during growth. Growing wild-type cells converted L-[U-14C]lysine into alpha-amino-[14C]adipate, suggesting both biosynthetic and degradative roles for alpha-aminoadipate. Lysine auxotrophs lys1, lys2 and lys3 of R. glutinis, unlike lysine auxotrophs of Saccharomyces cerevisiae, satisfied their growth requirement with L-pipecolate. Moreover, extracts of wild-type R. glutinis catalysed the conversion of L-pipecolate to alpha-aminoadipate-delta semialdehyde. These results suggest a biosynthetic role for L-pipecolate in R. glutinis but not in S. cerevisiae.     

Crystal structure of a lysine biosynthesis enzyme,LysX, from Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.     

Two unlinked lysine genes (LYS9 and LYS14) are required for the synthesis of saccharopine reductase in Saccharomyces cerevisiae.

Three lysine auxotrophs, strains AU363, 7305d, and 8201-7A, were investigated genetically and biochemically to determine their gene loci, biochemical lesions, and roles in the lysine biosynthesis of Saccharomyces cerevisiae. These mutants were leaky and blocked after the alpha-aminoadipate step. Complementation studies placed these three mutations into a single, new complementation group, lys14. Tetrad analysis from appropriate crosses provided evidence that the lys14 locus represented a single nuclear gene and that lys14 mutants were genetically distinct from the other mutants (lys1, lys2, lys5, and lys9) blocked after the alpha-aminoadipate step. The lys14 strains, like lys9 mutants, accumulated alpha-aminoadipate-semialdehyde and lacked significant amounts of saccharopine reductase activity. On the bases of these results, it was concluded, therefore, that LYS9 and LYS14, two distinct genes, were required for the biosynthesis of saccharopine reductase in wild-type S. cerevisiae.     

Effect of Hydroxylysine on the Biosynthesis of Lysine in Saccharomyces

Hydroxylysine acts as a growth inhibitor of Saccharomyces for a certain period of time. The inhibition is concentration-dependent and is reversed by a small amount of lysine in the medium. After the growth-inhibitory period, the wild-type cells are able to grow rapidly even in the presence of hydroxylysine. Both lysine auxotrophs and wild-type cells are unable to utilize hydroxylysine in place of lysine. Hydroxylysine, mimicking lysine, controls the biosynthesis of lysine and thereby limits the availability of biosynthetic lysine to the cells. Hydroxylysine affects the biosynthesis of lysine at a number of enzymatic steps. Accumulation of homocitric acid, the first intermediate of lysine biosynthesis, in the mutant strains 19B and A B9 is reduced significantly in the presence of hydroxylysine. Hydroxylysine, like lysine, exerts a significant inhibition in vitro on the homocitric acid-synthesizing activity. Enzymes following the alpha-aminoadipic acid step respond in a noncoordinate fashion to hydroxylysine. Level of the enzyme saccharopine reductase, but not of alpha-aminoadipic acid reductase or saccharopine dehydrogenase, is reduced significantly. These regulatory effects of hydroxylysine are similar to those observed for lysine.     

Distribution of saccharopine and 2-aminoadipic acid in higher plants

Saccharopine and 2-aminoadipic acid have been identified in eleven plant species belonging to nine families. The amino acids have been isolated from green parts of the plants using ion-exchange chromatography and preparative high voltage electrophoresis, and in three cases the identification was supported by mass spectroscopy. Mild conditions were used during the isolation to avoid lactamization, and the contents of saccharopine and 2-aminoadipic acid have been determined semiquantitatively. The significance of the occurrence of the two amino acids with regard to lysine metabolism is briefly discussed.     

Lysine-ketoglutarate reductase in human tissues.

Lysine-ketoglutarate reductase (saccharopine dehydrogenase (NADP+, lysine-forming) EC 1.5.1.8) from human liver has been partially purified and characterized. A spectrophotometric assay is described. The Michaelis constants have been determined for lysine (1.5-10-3 M), alpha-ketoglutarate (1-10-3 M) and NADPH (8-10-5 M). The pH optimum is 7.8. The enzyme is product inhibited. The specificity of the enzyme, response to inhibitors, pH and thermal stability are reported. Lysine-ketoglutarate reductase is present in high concentration in liver and heart, to a lesser degree in kidney and skin and in trace amounts in several other tissues. Saccharopine dehydrogenase (saccharopine dehydrogenase (NAD+, L-glutamate-forming) EC 1.5.1.9) was demonstrable only in liver and kidney. Lysine-ketoglutarate reductase reacts effectively with delta-hydroxylysine.     

Crystal structure of Mycobacterium tuberculosis diaminopimelate decarboxylase,an essential enzyme in bacterial lysine biosynthesis

The Mycobacterium tuberculosis lysA gene encodes the enzyme meso-diaminopimelate decarboxylase (DAPDC), a pyridoxal-5'-phosphate (PLP)-dependent enzyme. The enzyme catalyzes the final step in the lysine biosynthetic pathway converting meso-diaminopimelic acid (DAP) to l-lysine. The lysA gene of M. tuberculosis H37Rv has been established as essential for bacterial survival in immunocompromised mice, demonstrating that de novo biosynthesis of lysine is essential for in vivo viability. Drugs targeted against DAPDC could be efficient anti-tuberculosis drugs, and the three-dimensional structure of DAPDC from M. tuberculosis complexed with reaction product lysine and the ternary complex with PLP and lysine in the active site has been determined. The first structure of a DAPDC confirms its classification as a fold type III PLP-dependent enzyme. The structure shows a stable 2-fold dimer in head-to-tail arrangement of a triose-phosphate isomerase (TIM) barrel-like alpha/beta domain and a C-terminal beta sheet domain, similar to the ornithine decarboxylase (ODC) fold family. PLP is covalently bound via an internal aldimine, and residues from both domains and both subunits contribute to the binding pocket. Comparison of the structure with eukaryotic ODCs, in particular with a di-fluoromethyl ornithine (DMFO)-bound ODC from Trypanosoma bruceii, indicates that corresponding DAP-analogues might be potential inhibitors for mycobacterial DAPDCs.     

Purification and Characterization of the Bifunctional Enzyme Lysine-Ketoglutarate Reductase-Saccharopine Dehydrogenase from Maize

The first enzyme of the lysine degradation pathway in maize (Zea mays L.), lysine-ketoglutarate reductase, condenses lysine and [alpha]-ketoglutarate into saccharopine using NADPH as a cofactor, whereas the second, saccharopine dehydrogenase, converts saccharopine to [alpha]-aminoadipic-[delta]-semialdehyde and glutamic acid using NAD+ or NADP+ as a cofactor. The reductase and dehydrogenase activities are optimal at pH 7.0 and 9.0, respectively. Both enzyme activities, co-purified on diethylaminoethyl-cellulose and gel filtration columns, were detected on nondenaturing polyacrylamide gels as single bands with identical electrophoretic mobilities and share tissue specificity for the endosperm. The highly purified preparation containing the reductase and dehydrogenase activities showed a single polypeptide band of 125 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native form of the enzyme is a dimer of 260 kD. Limited proteolysis with elastase indicated that lysine-ketoglutarate reductase and saccharopine dehydrogenase from maize endosperm are located in two functionally independent domains of a bifunctional polypeptide.     

The reaction of pyruvate with saccharopine dehydrogenase.

The preceding paper in this journal has reported that pyruvate could be substituted for 2-oxo-glutarate as a substrate of saccharopine dehydrogenase [epsilon-N-(L-glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine-forming) in the direction of reductive condensation. In the present communication, the kinetic mechanism of saccharopine dehydrogenase reaction with NADH, L-lysine and pyruvate as reactants is reported. The results of initial velocity study, inhibition studies with lysine analogs and a reaction product, NAD+, are consistent with an ordered mechanism with the coenzyme binding first and pyruvate last. The reaction mechanism is at variance with that of the normal reaction in which 2-oxoglutarate is the substrate, in that the order of addition of the amino and oxo acid substrates is reversed. This fact suggests that there exists a small degree of randomness in the binding of amino and oxo acid substrates. From a product inhibition study, NAD+ was shown to be the last reactant released. Saccharopine [epsilon-N-(L-glutaryl-2)-L-lysine] was found to act as a potent dead-end inhibitor of the condensation reactions (of lysine and 2-oxoglutarate, and of lysine and pyruvate) by forming an abortive E. NADH. saccharopine complex.     

Efficient biosynthesis of α-aminoadipic acid via lysine catabolism in Escherichia coli

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.     

Effects of Supersuppressor Genes on Enzymes Controlling Lysine Biosynthesis in Saccharomyces

Yeast supersuppressor genes capable of masking the effects of several lysine mutant genes (ly(1-1), ly(9-1), ly(2-1)) were studied with respect to their effects on the respective enzymes (saccharopine dehydrogenase, saccharopine reductase, and alpha-amino-adipic acid reductase). In all strains tested, the supersuppressors functioned by allowing enzyme synthesis not found in the unsuppressed mutant. Studies by optical methods of saccharopine dehydrogenase and saccharopine reductase extracted from suppressed ly(1-1) and ly(9-1) cells, respectively, revealed that the K(m) values for these enzymes were significantly greater than those found in wild type. Saccharopine dehydrogenase from suppressed ly(9-1) cells was found to have K(m) values similar to wild type. These findings are consistent with the inference that a supersuppressor may act by enabling nonsense codons to be read, producing altered enzyme protein. Recent findings that lysine degradation in mammals may involve saccharopine and that the human diseases, hyperlysinemia and saccharopinuria, may be due to metabolic blocks in this route of lysine degradation suggest the ly(1-1) and ly(9-1) yeast mutants as models for the human condition and its possible euphenic treatment.     

Overall kinetic mechanism of saccharopine dehydrogenase (L-glutamate forming) from Saccharomyces cerevisiae

Kinetic studies were carried out for histidine-tagged saccharopine reductase from Saccharomyces cerevisiae at pH 7.0, suggesting a sequential mechanism with ordered addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the free enzyme followed by L-alpha-aminoadipate-delta-semialdehyde ( L-AASA) which adds in rapid equilibrium prior to l-glutamate in the forward reaction direction. In the reverse reaction direction, nicotinamide adenine dinucleotide phosphate (NADP) adds to the enzyme followed by addition of saccharopine. Product inhibition by NADP is competitive vs NADPH and noncompetitive vs alpha-AASA and L-glutamate, suggesting that the dinucleotide adds to the free enzyme prior to the aldehyde. Saccharopine is noncompetitive vs NADPH, alpha-AASA, and L-glutamate. In the direction of saccharopine oxidation, NADPH is competitive vs NADP and noncompetitive vs saccharopine, L-glutamate is noncompetitive vs both NADP and saccharopine, while L-AASA is noncompetitive vs saccharopine and uncompetitive vs NADP. The sequential mechanism is also corroborated by dead-end inhibition studies using analogues of AASA, L-glutamate, and saccharopine. 2-Amino-6-heptenoic acid was chosen as a dead-end analogue of L-AASA and is competitive vs AASA, uncompetitive vs NADPH, and noncompetitive vs L-glutamate. alpha-Ketoglutarate (alpha-Kg) serves as the dead-end analogue of L-glutamate and is competitive vs L-glutamate and uncompetitive vs L-AASA and NADPH. In the direction of saccharopine oxidation, N-oxalylglycine, L-pipecolic acid, L-leucine, alpha-ketoglutarate, glyoxylic acid, and L-ornithine were used as dead-end analogues of saccharopine and showed competitive inhibition vs saccharopine and uncompetitive inhibition vs NADP. The equilibrium constant for the reaction was measured at pH 7.0 by monitoring the change in absorbance of NADPH and is 200 M(-1). The value is in good agreement with the value determined using the Haldane relationship.     

Novel Posttranslational Activation of the LYS2-Encoded α-Aminoadipate Reductase for Biosynthesis of Lysine and Site-Directed Mutational Analysis of Conserved Amino Acid Residues in the Activation Domain of Candida albicans

The alpha-aminoadipate pathway for lysine biosynthesis is present only in fungi. The alpha-aminoadipate reductase (AAR) of this pathway catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde by a complex mechanism involving two gene products, Lys2p and Lys5p. The LYS2 and LYS5 genes encode, respectively, a 155-kDa inactive AAR and a 30-kDa phosphopantetheinyl transferase (PPTase) which transfers a phosphopantetheinyl group from coenzyme A (CoA) to Lys2p for the activation of Lys2p and AAR activity. In the present investigation, we have confirmed the posttranslational activation of the 150-kDa Lys2p of Candida albicans, a pathogenic yeast, in the presence of CoA and C. albicans lys2 mutant (CLD2) extract as a source of PPTase (Lys5p). The recombinant Lys2p or CLD2 mutant extract exhibited no AAR activity with or without CoA. However, the recombinant 150-kDa Lys2p, when incubated with CLD2 extract and CoA, exhibited significant AAR activity compared to that of wild-type C. albicans CAI4 extract. The PPTase in the CLD2 extract was required only for the activation of Lys2p and not for AAR reaction. Site-directed mutational analysis of G882 and S884 of the Lys2p activation domain (LGGHSI) revealed no AAR activity, indicating that these two amino acids are essential for the activation. Replacement of other amino acid residues in the domain resulted in partial or full AAR activity. These results demonstrate the posttranslational activation and the requirement of specific amino acid residues in the activation domain of the AAR of C. albicans.     

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.