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3bxm

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Structure of an inactive mutant of human glutamate carboxypeptidase II [GCPII(E424A)] in complex with N-acetyl-Asp-Glu (NAAG)

Structural highlights

3bxm is a 2 chain structure with sequence from Homo sapiens. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.71Å
Ligands:	, , , , , ,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

FOLH1_HUMAN Has both folate hydrolase and N-acetylated-alpha-linked-acidic dipeptidase (NAALADase) activity. Has a preference for tri-alpha-glutamate peptides. In the intestine, required for the uptake of folate. In the brain, modulates excitatory neurotransmission through the hydrolysis of the neuropeptide, N-aceylaspartylglutamate (NAAG), thereby releasing glutamate. Isoform PSM-4 and isoform PSM-5 would appear to be physiologically irrelevant. Involved in prostate tumor progression. Also exhibits a dipeptidyl-peptidase IV type activity. In vitro, cleaves Gly-Pro-AMC.

Evolutionary Conservation

Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.

Publication Abstract from PubMed

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally, we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 A resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be DeltaG(++) approximately 22(+/-5) kcal x mol(-1), which is in a good agreement with the experimentally observed reaction rate constant (k(cat) approximately 1 s(-1)). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the atomic level.

Reaction mechanism of glutamate carboxypeptidase II revealed by mutagenesis, X-ray crystallography, and computational methods.,Klusak V, Barinka C, Plechanovova A, Mlcochova P, Konvalinka J, Rulisek L, Lubkowski J Biochemistry. 2009 May 19;48(19):4126-38. PMID:19301871^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Klusak V, Barinka C, Plechanovova A, Mlcochova P, Konvalinka J, Rulisek L, Lubkowski J. Reaction mechanism of glutamate carboxypeptidase II revealed by mutagenesis, X-ray crystallography, and computational methods. Biochemistry. 2009 May 19;48(19):4126-38. PMID:19301871 doi:10.1021/bi900220s