Chemical Rescue and Inhibition Studies to Determine the Role of Arg301 in Phosphite Dehydrogenase

Abstract

Phosphite dehydrogenase (PTDH) catalyzes the NAD+-dependent oxidation of phosphite to phosphate. This reaction requires the deprotonation of a water nucleophile for attack on phosphite. A crystal structure was recently solved that identified Arg301 as a potential base given its proximity and orientation to the substrates and a water molecule within the active site. Mutants of this residue showed its importance for efficient catalysis, with about a 100-fold loss in k cat and substantially increased K m,phosphite for the Ala mutant (R301A). The 2.35 Å resolution crystal structure of the R301A mutant with NAD+ bound shows that removal of the guanidine group renders the active site solvent exposed, suggesting the possibility of chemical rescue of activity. We show that the catalytic activity of this mutant is restored to near wild-type levels by the addition of exogenous guanidinium analogues; Brønsted analysis of the rates of chemical rescue suggests that protonation of the rescue reagent is complete in the transition state of the rate-limiting step. Kinetic isotope effects on the reaction in the presence of rescue agents show that hydride transfer remains at least partially rate-limiting, and inhibition experiments show that K i of sulfite with R301A is ∼400-fold increased compared to the parent enzyme, similar to the increase in K m for phosphite in this mutant. The results of our experiments indicate that Arg301 plays an important role in phosphite binding as well as catalysis, but that it is not likely to act as an active site base.

Investigation of the proton relay system operative in human cystosolic aminopeptidase P

“…We observed that the R535A mutant losses its activity to a greater extent than does the wild-type protein at the same hydrazine concentration ( Fig 3C ), a possible result of the lack of hydrogen bond formation caused by the missing guanidinium group in R535A. Interestingly, a similar phenomenon was observed in studies with phosphite dehydrogenase, in which the side chain of Arg301, protruding towards the active site, plays a crucial role in forming the phosphite binding entrance via water molecule mediated hydrogen bonding to Trp134 [ 50 ]. Likewise, other enzymes also utilize the arginine side chain for proper substrate binding [ 48 , 53 – 57 ].…”

“…Previous studies with carboxypeptidase, E . coli ornithine transcarbamylase, asparagine synthetase B, alkanesulfonate monooxygenase and phosphite dyhedrogenase have identified the participation of arginine in enzymatic catalysis by showing the existence of exogenous guanidine mediated activity restoration corresponding to an enhancement in k cat [ 34 , 47 – 50 ]. Among these, the respective R57G and R325A mutants of ornithine transcarbamylase and asparagine synthetase B also displayed preferences for rate enhancements (10% and 15%, respectively) by guanidine hydrochloride over other guanidinium based agents, which is similar to that of hcAMPP ( Fig 3B ) [ 34 , 48 ].…”

“…In support of the proposed Mn 1 Asp415… Arg535 … Tyr527 motif, the guanidine side chain of Arg535 is separated by 2.8Å from both side chains of the neighboring residues, forming the hydrogen bond network that facilitates proton transfer in dual directions. As observed in a variety of proton-relay systems, the guanidinium group might also play roles in transition state stabilization and proper substrate orientation for hydride transfer [ 48 , 50 , 58 ]. Alternatively, this network is found in essentially all X-proline-specific pita bread domain enzymes including hcAMPP.…”

New insights into the mechanism of substrates trafficking in Glyoxylate/Hydroxypyruvate reductases

“…A search for similar structures within the PDB using the server DALI 16 reveals that the secondary structure of GRHPR (of PfuGRHPR as well as PyaGRHPR) shares significant similarity with phosphite dehydrogenase (PTDH) especially with PTDH from Pseudomonas stutzeri 17 18 (PDB ID Code: 4E5N (binary complex) and 4E5K (ternary complex)). Indeed, the superposition of PstPTDH (4E5K) with PfuGRHPR or PyaGRHPR gives RMSD values of 1.32 Å and 1.29 Å with sequence identity only close to 34%.…”

“…In PfuGRHPR structure, this interaction is obviously abolished with glyoxylate. Despite a different substrate, phosphite dehydrogenase (PTDH) possesses a high structural similarity with GRHPR and the active-site residues are strictly conserved 17 18 . In PTDH, systematic mutations of the three residues composing the network (Arg297, Ser291 and Trp138) show a clear effect on substrate affinity 20 .…”