Conformation of the C-peptide fragment of RNase A is calculated by Monte Carlo simulated annealing. We adopt the total potential energy as given by the sum of generic interatomic energies whose parameters are determined separately for each amino acid without referring to the empirical structure of the C-peptide. The simulation is carried out in a completely unrestricted way without imposing any weight towards given final destinations. Starting from completely random initial conformations and minimizing the total potential energy with respect to main-chain dihedral angles and side-chain torsion angles, we have obtained partial alpha-helix structure with a high probability (approximately 40%). The energetically most favourable structure exhibits a 2.5-turn alpha-helix at the location identical with that of the 3-turn alpha-helix in the native enzyme molecule. Classification of conformations obtained in the simulation into clusters of similar structure shows that our simulation indeed predicts the alpha-helix structure for the isolated C-peptide with specific charged residues. The results of simulation with various amino acid substitutions are also found to be consistent with the experimental implication for the importance of intramolecular ionic interactions for alpha-helix stability for this peptide.

The imidazole ¹⁵N signals of histidine 64 (His⁶⁴), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with ¹⁵N at ring-N^δ1 and N^ϵ2, ¹³Cat ring-Cϵ1, ¹³C and ¹⁵N at all carbon and nitrogen, or ¹⁵N at the amide nitrogen and the labeled glycine with ¹³C at the carbonyl carbon. Using the pH dependence of ring-¹⁵N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K_T) of His⁶⁴ is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His⁶⁴ as both a general acid (a) and base (b): its ϵ2-nitrogen as (a) releases one proton into the bulk, whereas itsδ1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the δ1-nitrogen of His⁶⁴. These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His⁶⁴ can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

Nicotinamide (NM) phosphoribosyltransferase (NMPRTase) catalyzes the reaction of NM and 5′-phosphoribosyl-1′-pyrophosphate (PRPP) to form NM mononucleotide (NMN) and pyrophosphate (PPi) in the pathway of NAD-biosynthesis. Monitoring the ¹H and ³¹P NMR spectra of the reaction mixture, we found that this reaction is reversible as dictated by the equilibrium constant K = [NMN][PPi]/([NM][PRPP]) = 0.14, which agreed well with the ratio of second-order rate constants for forward and backward reactions, K = 0.16. The crystal structures of this enzyme in the free form and bound to NM and PRPP at the resolution of 2.0–2.2 Å were essentially identical to that of the complex with NMN, except for some variations that could facilitate the substitution reaction by fixing the nucleophile and the leaving group for the requisite inversion of configuration at the C1’ carbon of the ribose ring. In the active site near the C1’ atom of the bound PRPP or NMN, there was neither negatively charged group nor waterproof environment necessary to support the feasibility of a ribo-oxocarbocation intermediate inherent in the S_N1 mechanism. The structures and catalytic mechanism thus revealed are also discussed in connection with the multiple biological functions of NMPRTase.