Biomedical subjects
Gregory A Petsko
Publications and source records attributed to Gregory A Petsko.
The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease.
Mutations in DJ-1, a human gene with homologues in organisms from all kingdoms of life, have been shown to be associated with autosomal recessive, early onset Parkinson's disease (PARK7). We report here the three-dimensional structure of the DJ-1 protein, determined at a resolution of 1.1 A by x-ray crystallography. The chain fold of DJ-1 resembles those of a bacterial protein, PfpI, that has been annotated as a cysteine protease, and of a domain of a bacterial catalase whose role in the activity of that enzyme is uncertain. In contrast to PfpI, a hexameric protein whose oligomeric structure is essential for its putative proteolytic activity, DJ-1 is a dimer with completely different intersubunit contacts. The proposed catalytic triad of PfpI is absent from the corresponding region of the structure of DJ-1, and biochemical assays fail to detect any protease activity for purified DJ-1. A highly conserved cysteine residue, which is catalytically essential in homologues of DJ-1, shows an extreme sensitivity to radiation damage and may be subject to other forms of oxidative modification as well. The structure suggests that the loss of function caused by the Parkinson's-associated mutation L166P in DJ-1 is due to destabilization of the dimer interface. Taken together, the crystal structure of human DJ-1 plus other observations suggest the possible involvement of this protein in the cellular oxidative stress response and a general etiology of neurodegenerative diseases.
A new recruit for the army of the men of death.
The army of the men of death, in John Bunyan's memorable phrase, has a new recruit, and fear has a new face: a face wearing a surgical mask.
2.4 A resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor.
This paper reports the first structure of a member of the Kex2/furin family of eukaryotic pro-protein processing proteases, which cleave sites consisting of pairs or clusters of basic residues. Reported is the 2.4 A resolution crystal structure of the two-domain protein ssKex2 in complex with an Ac-Ala-Lys-boroArg inhibitor (R = 20.9%, R(free) = 24.5%). The Kex2 proteolytic domain is similar in its global fold to the subtilisin-like superfamily of degradative proteases. Analysis of the complex provides a structural basis for the extreme selectivity of this enzyme family that has evolved from a nonspecific subtilisin-like ancestor. The P-domain of ssKex2 has a novel jelly roll like fold consisting of nine beta strands and may potentially be involved, along with the buried Ca(2+) ion, in creating the highly determined binding site for P(1) arginine.
War and peace.
If the fiftieth anniversary of the double helix teaches us anything it ought to be that great scientific discoveries are almost never made in time of war or directly for the purpose of war.
Galactose mutarotase: pH dependence of enzymatic mutarotation.
Here we report pH dependence of kinetic parameters for the mutarotation of alpha-D-glucose catalyzed by galactose mutarotase (GalM) from Escherichia coli. The values of k(cat) and k(cat)/K(m) for the mutarotation of alpha-D-galactose were found to be 1.84 x 10(4) s(-1) and 4.6 x 10(6) M(-1) s(-1), respectively, at pH 7.0 and 27 degrees C. The corresponding values for alpha-D-glucose were 1.9 x 10(4) s(-1) and 5.0 x 10(5) M(-1) s(-1). Inasmuch as the value of k(cat)/K(m) for the reaction of alpha-D-galactose is 10 times that for alpha-D-glucose, and the diffusional rate constants should be essentially the same for the two sugars, the mutarotation of alpha-D-glucose should not be diffusion controlled. Therefore, pH-rate profiles should not be distorted by diffusion. The k(cat) for the mutarotation of alpha-D-glucose is independent of pH. Therefore, either the enzyme-substrate complexes do not undergo ionization of catalytic groups, or the rate-limiting step is neither mutarotation nor diffusion. The profile of log k(cat)/K(m) versus pH is a distorted bell-shaped curve, with slopes of +1 on the acid side and -2 on the alkaline side. The values of pK(a) are 6.0 and 7.5, and mutarotation depends on the ionization states of three functional groups in the free enzyme, one unprotonated and two protonated. On the acid side, ring opening of alpha-D-glucose limits the rate, and on the alkaline side, ring closure of the open-chain sugar limits the rate. A mutarotation mechanism is presented in which one of the catalytic groups shuttles a proton to and from the endocyclic oxygen and the other two shuttle protons to the anomeric oxygen atoms. In this mechanism, three catalytic groups overcome the problem of nonstereospecificity in mutarotation. The groups are postulated to be His 104, His 175, and Glu 309. Mutations of these residues grossly impair catalytic activity. Variants H104Q- and E309Q-GalM display sufficient activity to allow profiles of log k(cat)/K(m) versus pH to be constructed. Both profiles show breaks on the acid side corresponding to pK(a) values of 5.8 for H104Q and 6.3 for E309Q. Apparently, ring opening of alpha-D-glucose limits the rate at low pHs, but ring closure does not become rate limiting at pHs up to 8.5 in reactions of these variants.
Still no flying cars.
Why, after less than two centuries of almost incomprehensible technological progress has progress in so many areas seemed to slow almost to a halt.
The immunosuppressive agent mizoribine monophosphate forms a transition state analogue complex with inosine monophosphate dehydrogenase.
Mizoribine monophosphate (MZP) is the active metabolite of the immunosuppressive agent mizoribine and a potent inhibitor of IMP dehydrogenase (IMPDH). This enzyme catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD via a covalent intermediate at Cys319 (E-XMP). Surprisingly, mutational analysis indicates that MZP is a transition state analogue although its structure does not resemble that of the expected transition state. Here we report the X-ray crystal structure of the E.MZP complex at 2.0 A resolution that reveals a transition state-like structure and solves the mechanistic puzzle of the IMPDH reaction. The protein assumes a new conformation where a flap folds into the NAD site and MZP, Cys319, and a water molecule are arranged in a geometry resembling the transition state. The water appears to be activated by interactions with a conserved Arg418-Tyr419 dyad. Mutagenesis experiments confirm that this new closed conformation is required for the hydrolysis of E-XMP, but not for the reduction of NAD. The closed conformation provides a structural explanation for the differences in drug selectivity and catalytic efficiency of IMPDH isozymes.
Funky, not junky.
In many respects, the most intriguing part of the mouse genome sequence is what it has told us about so-called 'junk' DNA sequences.
Everything I need to know about genomics, I learned from Yogi Berra.
As 2002 draws to a close, I find myself contemplating the future of biology with hope, but also with uncertainty and some apprehension. When I find myself in such a spiritual quandary, I usually turn to a guru.
Crystal structure of a ternary complex of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: NAD+ orients the active site loop for catalysis.
Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to XMP with the reduction of NAD(+), which is the rate-limiting step in the biosynthesis of guanine nucleotides. IMPDH is a promising target for chemotherapy. Microbial IMPDHs differ from mammalian enzymes in their lower affinity for inhibitors such as mycophenolic acid (MPA) and thiazole-4-carboxamide adenine dinucleotide (TAD). Part of this resistance is determined by the coupling between nicotinamide and adenosine subsites in the NAD(+) binding site that is postulated to involve an active site flap. To understand the structural basis of the drug selectivity, we solved the X-ray crystal structure of the catalytic core domain of Tritrichomonas foetus IMPDH in complex with IMP and beta-methylene-TAD at 2.2 A resolution. Unlike previous structures of this enzyme, the active site loop is ordered in this complex, and the catalytic Cys319 is 3.6 A from IMP, in the same plane as the hypoxanthine ring. The active site loop forms hydrogen bonds to the carboxamide of beta-Me-TAD which suggests that NAD(+) promotes the nucleophillic attack of Cys319 on IMP. The interactions of the adenosine end of TAD are very different from those in the human enzyme, suggesting the NAD(+) site may be an exploitable target for the design of antimicrobial drugs. In addition, a new K(+) site is observed at the subunit interface. This site is adjacent to beta-Me-TAD, consistent with the link between the K(+) activation and NAD(+). However, contrary to the coupling model, the flap does not cover the adenosine subsite and remains largely disordered.
The guards themselves.
If we agree that, in the present climate of fear of bioterrorism, some restrictions on the conduct and/or publication of certain types of biological research are likely, it is to our advantage to preempt government action by devising for ourselves restrictions that we can live with, then the inevitable question becomes: how should these restrictions be administered?
An Asilomar moment.
As governments in the US and Europe contemplate legislation that would divert funding of some genomics-driven research to Offices of Homeland Security and the like, and that would restrict the freedom of biologists to publish and share some of their data, we of the scientific community are facing a crisis, brought on by fears of bioterrorism, that eerily mirrors the early days of recombinant DNA research.
Misfolded proteins are competent to mediate a subset of the responses to heat shock in Saccharomyces cerevisiae.
Cells may sense heat shock via the accumulation of thermally misfolded proteins. To explore this possibility, we determined the effect of protein misfolding on gene expression in the absence of temperature changes. The imino acid analog azetidine-2-carboxylic acid (AZC) is incorporated into protein competitively with proline and causes reduced thermal stability or misfolding. We found that adding AZC to yeast at sublethal concentrations sufficient to arrest proliferation selectively induced expression of heat shock factor-regulated genes to a maximum of 27-fold and that these inductions were dependent on heat shock factor. AZC treatment also selectively repressed expression of the ribosomal protein genes, another heat shock factor-dependent process, to a maximum of 20-fold. AZC treatment thus strongly and selectively activates heat shock factor. AZC treatment causes this activation by misfolding proteins. Induction of HSP42 by AZC treatment required protein synthesis; treatment with ethanol, which can also misfold proteins, activated heat shock factor, but treatment with canavanine, an arginine analog less potent than AZC at misfolding proteins, did not. However, misfolded proteins did not strongly induce the stress response element regulon. We conclude that misfolded proteins are competent to specifically trigger activation of heat shock factor in response to heat shock.
Fish tale.
The draft sequence of the genome of the Japanese pufferfish has just been announced and the temptation to humor is great.