PDBsum entry 1sd0

Transferase

PDB id: 1sd0

Contents

Protein chain

356 a.a. *

Ligands

NO3

ARG

ADP

Metals

_MG

_CL

Waters ×288

* Residue conservation analysis

References listed in PDB file

Key reference

Title

The active site cysteine of arginine kinase: structural and functional analysis of partially active mutants.

Authors

J.L.Gattis, E.Ruben, M.O.Fenley, W.R.Ellington, M.S.Chapman.

Ref.

Biochemistry, 2004, 43, 8680-8689. [DOI no: 10.1021/bi049793i]

PubMed id

15236576

Abstract

Arginine kinase buffers cellular ATP levels by catalyzing reversible phosphoryl transfer between ATP and arginine. A conserved cysteine has long been thought important in catalysis. Here, cysteine 271 of horseshoe crab arginine kinase has been mutated to serine, alanine, asparagine, or aspartate. Catalytic turnover rates were 0.02-1.0% of wild type, but the activity of uncharged mutations could be partially rescued with chloride. Steady-state binding constants were slightly increased, more so for phospho-L-arginine than ADP. Substrate binding synergy observed in many phosphagen kinases was reduced or eliminated in mutant enzymes. The crystallographic structure of the alanine mutant at 2.3 A resolution, determined as a transition state analogue complex with arginine, nitrate, and MgADP, was nearly identical to wild type. Enzyme-substrate interactions are maintained as in wild type, and substrates remain at least roughly aligned for in-line phosphoryl transfer. Homology models with serine, asparagine, or aspartate replacing the active site cysteine similarly show only minor structural changes. Most striking, however, is the presence in the C271A mutant crystallographic structure of a chloride ion within 3.5 A of the nonreactive N(eta) substrate nitrogen, approximating the position of the sulfur in the wild-type's cysteine. Together, the results contradict prevailing speculation that the cysteine mediates a substrate-induced conformational change, confirm that it is the thiolate form that is relevant to catalysis, and suggest that one of its roles is to help to enhance the catalytic rate through electrostatic stabilization of the transition state.

Secondary reference #1

Title

Transition state structure of arginine kinase: implications for catalysis of bimolecular reactions.

Authors

G.Zhou, T.Somasundaram, E.Blanc, G.Parthasarathy, W.R.Ellington, M.S.Chapman.

Ref.

Proc Natl Acad Sci U S A, 1998, 95, 8449-8454. [DOI no: 10.1073/pnas.95.15.8449]

PubMed id

9671698

Figure 2.
Fig. 2. Details of the active site. For clarity only atoms in the immediate neighborhood are shown with carbon-colored black, oxygen red, nitrogen dark blue, magnesium light blue, sulfur yellow and phosphorus gray. Distances are shown in Å. (a) Stereo diagram comparing part of the experimental analog structure with omit-map electron density and the structure of the presumptive transition state (gray atoms). Small molecule model systems suggest a preassociative concerted phosphoryl transfer and a pentavalent -phosphorus transition state with about 20% covalent bonding to both the -phosphoryl oxygen and guanidino nitrogen (32, 33). The transition-state coordinates were derived from the experimental coordinates by replacing the nitrate with a phosphoryl group, and refining with additional distance and angle restraints appropriate for the estimated 20% partial covalent bonding. (b-e) Details of the enzyme-substrate analog interactions: (b) and phosphoryl groups of the ADP are held in place by extensive hydrogen bonds/salt bridges with four highly conserved arginines; (c) the nitrate (mimicking a planar phosphoryl group during transfer) is sandwiched between two conserved arginines and the Mg2+ ion whose position is constrained by ligands from the and phosphoryl groups of the ADP; (d) the guanidinium of the substrate arginine is clamped with salt bridges/hydrogen bonds to two carboxylates and a conserved cysteine that likely exists as a thiolate (54); and (e) interactions of the substrate amino and carboxylate groups with loop residues 63-68 of the enzyme. The carboxylate-to-backbone interactions might be conserved between all phosphagens and their kinases. The amino groups are present in arginine and lombricine but absent from creatine and glycocyamine. The tyrosine interacting with the amino group is conserved among AKs, but is a valine in all other phosphagen kinases. Immediately preceding residue 61 (and interactions with the carboxylate) is an insertion in other sequences whose size inversely correlates with the size of substrate (42).

Figure 3.
Fig. 3. Roles of neighboring amino acids in the catalytic mechanism of AK: (a) the forward reaction and (b) the reverse reaction. In this schematic representation, only the reactive groups of participants are shown. *, The structure does not indicate whether it is Glu-225 or Glu-314 that acts as the proposed acid/base catalyst.

Secondary reference #2

Title

Refinement of the arginine kinase transition-State analogue complex at 1.2 a resolution: mechanistic insights.

Authors

M.S.Yousef, F.Fabiola, J.L.Gattis, T.Somasundaram, M.S.Chapman.

Ref.

Acta Crystallogr D Biol Crystallogr, 2002, 58, 2009-2017. [DOI no: 10.1107/S0907444902014683]

PubMed id

12454458

Figure 2.
Figure 2 (a) Precise alignment of the substrates in the active site as revealed by the 2F[o] - F[c] electron-density map contoured at 3 . (b) The in-line transfer of the phosphoryl group and the hydrogen-bond geometry between O[ 1] of ADP and O[1] of nitrate. Angles in parentheses correspond to the optimal reaction trajectory (Zhou et al., 1998[Zhou, G., Somasundaram, T., Blanc, E., Parthsarathy, G., Ellington, W. R. & Chapman, M. S. (1998). Proc. Natl Acad. Sci. USA, 95, 8449-8454.]).

Figure 4.
Figure 4 Coordination of Mg2+ in the active site. Distances are shown in Å.

The above figures are reproduced from the cited reference with permission from the IUCr

Secondary reference #3

Title

Induced fit in guanidino kinases--Comparison of substrate-Free and transition state analog structures of arginine kinase.

Authors

M.S.Yousef, S.A.Clark, P.K.Pruett, T.Somasundaram, W.R.Ellington, M.S.Chapman.

Ref.

Protein Sci, 2003, 12, 103-111. [DOI no: 10.1110/ps.0226303]

PubMed id

12493833

Figure 1.
Figure 1. Example electron density: a 2F[o] - F[c] omit map, contoured at 1.5 , around residues Phe[218], Leu[219], and Val[220].

Figure 3.
Figure 3. Space-filling models comparing open and closed forms of arginine kinase. (A) The substrate-free conformation is shown with the substrates in stick model as they would be bound in the closed form. (B) The closed form shields the substrates from solvent access.

The above figures are reproduced from the cited reference with permission from the Protein Society