Tryptophan Synthase Mutations That Alter Cofactor Chemistry Lead to Mechanism-Based Inactivation

Abstract

Mutations in the pyridoxal phosphate binding site of the tryptophan synthase beta subunit (S377D and S377E) alter cofactor chemistry [Jhee, K.-H., et al. (1998) J. Biol. Chem. 273, 11417-11422]. We now report that the S377D, S377E, and S377A beta2 subunits form alpha2 beta2 complexes with the alpha subunit and activate the alpha subunit-catalyzed cleavage of indole 3-glycerol phosphate. The apparent Kd for dissociation of the alpha and beta subunits is unaffected by the S377A mutation but is increased up to 500-fold by the S377D and S377E mutations. Although the three mutant alpha2 beta2 complexes exhibit very low activities in beta elimination and beta replacement reactions catalyzed at the beta site in the presence of Na+, the activities and spectroscopic properties of the S377A alpha2 beta2 complex are partially repaired by addition of Cs+. The S377D and S377E alpha2 beta2 complexes, unlike the wild-type and S377A alpha2 beta2 complexes and the mutant beta2 subunits, undergo irreversible substrate-induced inactivation by L-serine or by beta-chloro-L-alanine. The rates of inactivation (kinact) are similar to the rates of catalysis (kcat). The partition ratios are very low (kcat/kinact = 0.25-3) and are affected by alpha subunit ligands and monovalent cations. The inactivation product released by alkali was shown by HPLC and by fluorescence, absorption, and mass spectroscopy to be identical to a compound previously synthesized from pyridoxal phosphate and pyruvate. We suggest that alterations in the cofactor chemistry that result from the engineered Asp377 in the active site of the beta subunit may promote the mechanism-based inactivation.

Human Cystathionine β-Synthase Is a Heme Sensor Protein. Evidence That the Redox Sensor Is Heme and Not the Vicinal Cysteines in the CXXC Motif Seen in the Crystal Structure of the Truncated Enzyme,

“…In this class of PLP enzymes, a polar residue (serine or threonine) is always found within hydrogen-bonding distance to the pyridinium nitrogen, either close to the back (si face) or the front (re face) of the cofactor, except in aminocyclopropane deaminase, where a glutamate is found directly below the PLP (27). Threonine synthase departs from this trend as a histidine is located on the re face.…”

Structural and Enzymatic Properties of 1-Aminocyclopropane-1-carboxylate Deaminase Homologue from Pyrococcus horikoshii

“…The charge density of the pyridine ring is strictly modulated by various factors in the neighborhood of the pyridine nitrogen atom in the PLP enzyme. [18][19][20][21] The pyridine nitrogen atom of hACCD exists within hydrogen bond distance (2.5 Å ) of the side-chain carboxyl oxygen atom of Glu296, which is not the case for other members of the TRPSb family. The crystallographically analyzed enzymes of this family are TRPSb, 22 O-acetylserine sulfhydrylase (cysteine synthase), 23 threonine deaminase, 24 threonine synthase, 25 and cystathionine b-synthase.…”

Identification of key amino acid residues in the catalytic mechanism of diaminopropionate ammonialyase from Salmonella typhimurium

“…There was no significant change in the catalytic activity with DL-DAP as substrate (Table 1) with both mutant enzymes, as observed in the case of OASS [19]. Mutation of the corresponding residue in the enzymes which follow the E 1 CB mechanism of b-elimination (TrpS and CBS) resulted in complete loss of activity [20,21]. These results suggested that the residue hydrogen bonded to the pyridine nitrogen is not crucial for b-elimination activity by DAPAL and might follow the anti-E 2 mechanism, as proposed for OASS [19].…”

“…). Such a pH‐dependent change in the absorbance spectrum was observed with the S377D mutant of TrpS β‐subunit which follows the E 1 CB mechanism . Mutation of Ser289 in yeast CBS to Ala or Asp resulted in a drastic reduction in β‐replacement activity and the fluorescence resonance energy transfer between the tryptophan residues of the enzyme and PLP was diminished or absent in the mutants, demonstrating that the Ser289 was essential for maintaining proper orientation of PLP.…”

“…The amino acid residue that interacts with N1 of PLP in most fold‐type II PLP‐dependent enzymes is a Ser and in fold‐type I PLP‐dependent enzymes it is an Asp. Mutational analysis of this crucial residue in TrpS, OASS and CBS resulted in the elucidation of their mechanism of catalysis . Unlike these enzymes, the residue involved in interaction with N1 of PLP was identified as Thr376 in eDAPAL, which is also conserved in sDAPAL (Thr385).…”