Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4′ hydrogen transfer on the coenzyme (original) (raw)
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Protein Science, 1995
Of the major amino acid side chains that anchor pyridoxal 5"phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, Le., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates. However, 0-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.
Analytical Biochemistry, 1997
does not interfere with the assay. The kinetic parameters determined for the transamination of phenylala-A continuous assay for Escherichia coli tyrosine aminine by TATase (k cat Å 180 s 01 , K M (L-Phe) Å 0.56 mM, K M notransferase (TATase) that employs Lactobacillus (a-KG) Å 5 mM) with HO-HxoDH as a coupling enzyme are delbrueckii ssp. bulgaricus hydroxyisocaproate dehycomparable to those reported in the literature, which drogenase (HO-HxoDH) as a coupling enzyme is dewere determined by direct monitoring of the formascribed. a-Keto acids, including those formed by tion of phenylpyruvate at 280 nm. This new assay of-TATase-catalyzed transamination of L-phenylalanine, fers the advantages of increased sensitivity and broad L-tyrosine, L-tryptophan, L-methionine, and L-leucine, substrate specificity. ᭧ 1997 Academic Press are converted to the corresponding a-hydroxy acids by the auxiliary enzyme. The concomitant reduction of NADH by this enzyme can be followed as a decrease
Biochemistry, 1996
The crystal structure of dimeric bacterial D-amino acid transaminase shows that the indole rings of the two Trp-139 side chains face each other in the subunit interface about 10 Å from the coenzyme, pyridoxal 5′-phosphate. To determine whether it has a role in the catalytic efficiency of the enzyme or interacts with the coenzyme, Trp-139 has been substituted by several different types of amino acids, and the properties of these recombinant mutant enzymes have been compared to the wild-type enzyme. In the native wild-type holoenzyme, the fluorescence of one of the three Trp residues per monomer is almost completely quenched, probably due to its interaction with PLP since in the native wild-type apoenzyme devoid of PLP, tryptophan fluorescence is not quenched. Upon reconstitution of this apoenzyme with PLP, the tryptophan fluorescence is quenched to about the same extent as it is in the native wild-type enzyme. The site of fluorescence quenching is Trp-139 since the W139F mutant in which Trp-139 is replaced by Phe has about the same amount of fluorescence as the wild-type enzyme. The circular dichroism spectra of the holo and the apo forms of both the wild-type and the W139F enzymes in the far-ultraviolet show about the same degree of ellipticity, consistent with the absence of extensive global changes in protein structure. Furthermore, comparison of the circular dichroism spectrum of the W139F enzyme at 280 nm with the corresponding spectral region of the wild-type enzyme suggests a restricted microenvironment for Trp-139 in the latter enzyme. The functional importance of Trp-139 is also demonstrated by the finding that its replacement by Phe, His, Pro, or Ala gives mutant enzymes that are optimally active at temperatures below that of the wild-type enzyme and undergo the E-PLP f E-PMP transition as a function of D-Ala concentration with reduced efficiency. The results suggest that a fully functional dimeric interface with the two juxtaposed indole rings of Trp-139 is important for optimal catalytic function and maximum thermostability of the enzyme and, furthermore, that there might be energy transfer between Trp-139 and coenzyme PLP.
The regulation of aminotransferase activity by carbamoyl-phosphate
Life Sciences, 1994
We studied the effect of carbamoylphosphate (CP) on L-aspartate aminotransferase (GOT) and L-alanine aminotransferase (GPT), compared to its effect on L-threonine deaminase (TD). GPT and GOT were slightly inhibited by CP, while TD was strongly inhibited. GPT and TD, but not GOT, were inactivated when preincubated with CP. Only GOT was enhanced by pyridoxal 5'-phosphate (PLP), but not when the coenzyme was preincubated with CP. When the enzymes were resolved by p-chloromercuribenzoate (PCMB) treatment to apoenzymes, only GOT retained 47% of the; original activity. Reeonstitution of the apoenzymes with PLP also followed different course; activities of GPT and TD were completely restored while GO[I' remained partially inactivated. Treatment of apoenzymes with CP resulted in impairment of their reconstitution except GPT, activity of which could be completely restored. When PLP was pre-treated with CP before reconstitution, however, even GPT was only partially restored. The data indicated that CP affect activities of these enzymes at different levels, holoenzymes, PLP and probably apoenzymes. Under a concentration of PLP, activity of GOT would be most enhanced, followed by TD then GPT. In the presence of CP, this effect would be eliminated.
Journal of Food Biochemistry, 1998
Two oligomeric species of an aromatic amino acid aminotransferase (Ar-AT) were purified and characterized from Lactococcus lactis subsp. lactis S3. The more abundant species, Ar-AT1, was purified over 2,200-fold with a 3% recovery. The molecular masses of Ar-AT1 were determined to be 42 kDa and 84 kDa by SDS-PAGE and gel filtration, respectively. The molecular masses of the less abundant species, Ar-AT2, were 42 kDa and 170 kDa, respectively. Both Ar-AT1 and Ar-AT2 have identical N-terminal sequences which indicates they are homodimeric and homotetrameric forms of the same enzyme. The Ar-ATs catalyze pyridoxal-5′-phosphate dependent transamination of Phe, Tyr, Trp, Leu and Met utilizing α-ketoglutarate as the amino acceptor. Km values of the two Ar-ATs for Trp, Tyr and Phe ranged from 0.65 to 2.43 mM. However, differences were observed in the pI and specific activities between the two Ar-ATs. The pI values of Ar-AT1 and Ar-AT2 were 4.63 and 3.93, respectively. The Vmax/Km ratios of Ar-AT2 for Trp, Tyr and Phe were three-fold greater than that of Ar-AT1, indicating that Ar-AT2 is more catalytically efficient on these amino acids than Ar-AT1.
Crystal Structure of a D-Amino Acid Aminotransferase: How the Protein Controls Stereoselectivity
Biochemistry, 1995
The three-dimensional structure of D-amino acid aminotransferase (D-AAT) in the pyridoxamine phosphate form has been determined crystallographically. The fold of this pyridoxal phosphate (PLP)containing enzyme is completely different from those of any of the other enzymes that utilize PLP as part of their mechanism and whose structures are known. However, there are some striking similarities between the active sites of D-AAT and the corresponding enzyme that transaminates L-amino acids, L-aspartate aminotransferase. These similarities represent convergent evolution to a common solution of the problem of enforcing transamination chemistry on the PLP cofactor. Implications of these similarities are discussed in terms of their possible roles in the stabilization of intermediates of a transamination reaction. In addition, sequence similarity between D-AAT and branched chain L-amino acid aminotransferase suggests that this latter enzyme will also have a fold similar to that of D-AAT. / LY1145 k kelimmc pyridorminx md PYNVB(C FIGURE 1: Reaction catalyzed by the D-aminO acid aminotransferase. The cofactor is shown positioned as seen in the structure of D-AAT with the side facing solvent toward the viewer and the re side facing the protein. The incoming a-amino acid reacts with the internal aldimine between the active site lysine and pyridoxal phosphate (PLP) to give an external aldimine between the amino